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Proteomic Characterization of Postmortem Amyloid Plaques Isolated by Laser Capture Microdissection

Proteomic Characterization of Postmortem Amyloid Plaques Isolated by Laser Capture Microdissection THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 35, Issue of August 27, pp. 37061–37068, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Proteomic Characterization of Postmortem Amyloid Plaques □ S Isolated by Laser Capture Microdissection* Received for publication, April 2, 2004, and in revised form, June 24, 2004 Published, JBC Papers in Press, June 25, 2004, DOI 10.1074/jbc.M403672200 Lujian Liao‡§, Dongmei Cheng‡§, Jian Wang‡§, Duc M. Duong‡§, Tatyana G. Losik‡§, Marla Gearing§ , Howard D. Rees§ , James J. Lah§ , Allan I. Levey§ , and Junmin Peng‡§** From the ‡Department of Human Genetics, §Center for Neurodegenerative Disease, the Department of Pathology and Laboratory Medicine, and the Department of Neurology, Emory University School of Medicine, Atlanta, Georgia 30322 The first major breakthrough in understanding the molecu- The presence of amyloid plaques in the brain is one of the pathological hallmarks of Alzheimer’s disease (AD). lar pathogenesis of AD came from the biochemical purification We report here a comprehensive proteomic analysis of of amyloid -peptide (A) from senile plaques, as described by senile plaques from postmortem AD brain tissues. Senile Glenner and Wong (6), and the subsequent sequencing and plaques labeled with thioflavin-S were procured by la- identification of the A precursor protein (APP) gene. Although ser capture microdissection, and their protein compo- the major insoluble component of plaques has been identified nents were analyzed by liquid chromatography coupled as A (6, 7), the entire molecular composition of the plaques is with tandem mass spectrometry. We identified a total of not known. The plaques are highly complex structures with a 488 proteins coisolated with the plaques, and we found variety of neural and glial elements (8), and many proteins multiple phosphorylation sites on the neurofilament in- have been localized to these structures by immunohistochem- termediate chain, implicating the complexity and diver- istry. However, biochemical verification of the plaque compo- sity of cellular processes involved in the plaque forma- nents has been scarce. Moreover, biochemical approaches pre- tion. More significantly, we identified 26 proteins viously applied to purify plaque components generally use very enriched in the plaques of two AD cases by quantitative stringent extraction conditions (e.g. high concentration of salt, comparison with surrounding non-plaque tissues. The urea, and/or protease treatment) that may remove A-associ- localization of several proteins in the plaques was fur- ther confirmed by the approach of immunohistochemis- ated proteins. The identities and roles of other plaque proteins try. In addition to previously identified plaque constit- that may act synergistically or competitively with A in aggre- uents, we discovered novel association of dynein heavy gation and deposition are also incomplete. A systematic anal- chain with the plaques in human postmortem brain and ysis of plaque proteins should shed light on the underlying in a double transgenic AD mouse model, suggesting that molecular processes that govern the plaque formation. neuronal transport may play a role in neuritic degener- Traditionally, proteomic analysis is performed by comparing ation. Overall, our results revealed for the first time the samples between AD cases and normal controls in two-dimen- sub-proteome of amyloid plaques that is important for sional gels and identifying proteins of interest by mass spec- further studies on disease biomarker identification and trometry (9). Several groups (10, 11) have tried this strategy molecular mechanisms of AD pathogenesis. and identified some proteins that are altered in the expression levels. However, the two-dimensional gel method is generally 1 incompatible with proteins of extreme size, pI, and/or hydro- Alzheimer’s disease (AD) is a devastating neurological dis- phobicity, and it is tedious to determine the identity of hun- order that impairs cognitive function and disturbs emotion and dreds of protein spots displayed on a two-dimensional gel. personality. Histopathologically, AD is manifested by the ex- Many of the limitations can be overcome by the development of tracellular aggregation of amyloid plaques and the intraneuro- liquid chromatography combined with tandem mass spectrom- nal neurofibrillary tangles. Although current amyloid cascade etry (LC-MS/MS) (12, 13). It is now possible to analyze hun- hypothesis (1) or tau hypothesis (2) provides a framework for dreds to thousands of proteins directly from a complex protein studying AD pathogenesis, the detailed molecular mechanisms mixture, and the sensitivity can reach low femtomole and even that translate amyloid or tau accumulation into neuronal dam- subfemtomole levels. The power of shotgun LC-MS/MS-based age and functional brain impairments are largely unknown. In proteomics technology has been documented by successful pro- addition, there are numerous, complex pathological changes in teomic analysis of subcellular structures in mammalian cells, AD brain that contribute to neuronal and synaptic degenera- such as nucleolus (14), centrosome (15), nuclear envelope (16), tion, including mitochondrial dysfunction, oxidative damage, and inflammation (3–5). mitochondria (17), and postsynaptic density (18). The method- ology has also been used to address dynamic changes in protein levels under specific conditions, as exemplified by the identifi- * The costs of publication of this article were defrayed in part by the cation of protein components in the epidermal growth factor payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to signaling pathway (19) and the functional investigation of the indicate this fact. myc oncogene (20). Mass spectrometry has also greatly simpli- □ S The on-line version of this article (available at http://www.jbc.org) fied the analysis of post-translational modifications (21, 22). contains Table S1. ** To whom correspondence should be addressed. E-mail: jpeng@ Whereas it may be problematic to isolate plaques to homoge- genetics.emory.edu. neity by using traditional biochemical methods, the advent of The abbreviations used are: AD, Alzheimer’s disease; LC-MS/MS, laser capture microdissection (LCM) allows procuring a micro- liquid chromatography coupled with tandem mass spectrometry; LCM, scopic region as small as 3–5 m in diameter (23). The LCM laser capture microdissection; A, amyloid -peptide; APP, A precur- sor protein. technology has been used extensively to collect homogeneous cell This paper is available on line at http://www.jbc.org 37061 This is an Open Access article under the CC BY license. 37062 Proteomic Characterization of Amyloid Plaques Database Searching for Protein Identification—The Sequest algo- types for DNA mutation detection and gene expression analysis rithm (30) was utilized for searching all MS/MS spectra against the at the mRNA level (23). More recently, its application in proteom- human reference data base (ftp.ncbi.nih.gov/genbank, July, 2003). The ics has begun to be appreciated (24, 25). It is possible to use LCM parameters were set to allow parent ion mass tolerance to be three and to capture neuropathological structures with high purity, but the to consider only the b and y ion series. Modifications were permitted to amount of samples collected by LCM is often limited, complicat- allow the detection of the following (mass shift shown in daltons): ing proteomic analysis by two-dimensional gel-based methods. oxidized methionine (16), carboxymethylated cysteine (57), and phosphorylated serine, threonine, and tyrosine (80). We used more These limitations can be alleviated by the integration of LCM stringent Sequest criteria than described previously (31, 32): 1) only with the highly sensitive LC-MS/MS technology. fully tryptic peptides were considered; 2) Cn score is at least 0.08; and Here we present the isolation of postmortem amyloid plaques 3) Xcorr should be larger than 2.0, 1.7, or 3.3 for charge states of 1, 2, labeled with thioflavin-S by using the LCM approach and the 3, respectively. To reduce false-positives further, we manually veri- systematic identification of extracted proteins by LC-MS/MS. fied proteins matched by less than three peptides, because no false- positives were found among proteins identified by at least three distinct The entire procedure was performed twice with samples from two peptides (32). Therefore, all peptides were accepted with high confi- AD cases. Together, more than 480 proteins were detected in the dence. The conversion from the identified peptides to proteins was plaque samples, and 26 proteins were shown to be enriched in the complicated by the presence of different names for the same protein plaques in comparison with the surrounding tissue. The presence and/or by the sharing of peptides within several proteins (e.g. protein of selected proteins in the plaques was confirmed by immunohis- paralogs) (33). Thus we manually verified all proteins and removed the redundancy. Typically, we accepted proteins identified by at least one tochemistry, and cytoplasmic dynein was found to be a novel “unique peptide.” Obvious contaminants such as trypsin and keratins component of amyloid plaques in human specimens and in an AD were removed. Finally we merged the data sets of the plaque samples mouse model. These studies demonstrate a powerful new ap- from two independent experiments. proach for achieving comprehensive analysis of the sub-proteome Protein Quantification by Mass Spectrometry—Quantitative protein of neuropathological structures. comparison between the plaques and the non-plaque control was car- ried out in two steps. The first step was based on the number of peptides MATERIALS AND METHODS identified for an assigned protein, indicative of protein abundance. We discarded proteins that were identified by more peptides in the control Brain Sections—Small blocks of fresh frontal and temporal cortex than in the plaques from either AD case. The second step was based on were obtained at autopsy from a 55-year-old Caucasian female (post- extracted ion current of corresponding peptides in MS survey scans (15, mortem interval, 4.5 h, case 1) and a 78-year-old Caucasian male 34, 35). The ratio of peak intensities of selected peptides was measured (postmortem interval, 17.5 h, case 2). Both cases were neuropathologi- in the 15 pairs of peptide mixtures that were generated by in-gel cally diagnosed as definite AD according to CERAD criteria (26). The digestion of the 15 pairs of gel pieces as shown (Fig. 1). We analyzed brain blocks were embedded in Tissue-Tek® OCT Compound (Jed Pella each pair of samples in two consecutive LC-MS/MS runs on the same Inc., Redding, CA), frozen on dry ice, and stored at 80 °C. 10-m-thick column and found that the quantitative variation was within 2-fold in brain sections were cut by using a Leica CM 3050 cryostat (Leica general by using a trypsin auto-cleavage peptide (VATVSLPR, m/z Microsystems Inc., Bannockburn, IL) and mounted on uncoated and 842.5 for singly charged ion) as internal control. Therefore, we used uncharged glass slides. 2-fold as the threshold for protein enrichment in the plaques. The Laser Capture Microdissection—Isolation of plaques by LCM was trypsin auto-cleavage peptide was also used to normalize the measured performed based on the protocol developed previously (27). The frozen peptide ratios and to normalize the elution time of selected peptides brain sections were thawed at room temperature, fixed with 75% eth- between the pair of LC-MS/MS analyses. It should be mentioned that, anol for 1 min, stained with 1% thioflavin-S for 1 min, differentiated in in contrast to the peptide identification that was primarily derived from 75% ethanol for 1 min, and then subjected to dehydration in a series of its MS/MS spectrum (Fig. 3B), the peptide quantification relied on the graded ethanol. Finally, the slides were cleared in xylene for 5 min and MS survey scan (Fig. 3A). An MS survey scan allowed the detection of then air-dried and desiccated. LCM was performed the same day under many peptide ion peaks, of which only the most three predominant were a fluorescence microscope attached to a Pixcell II laser capture facility selected for sequencing by MS/MS analysis. However, the other nonse- (Arcturus, Mountain View, CA) with the following settings: excitation quenced peaks on MS survey scans could be useful for quantification. wavelength, 495 nm; laser power, 60 – 80 milliwatts; duration, 1 ms; For example, when a peptide was sequenced by MS/MS analysis in the and laser spot size, 7.5 m. A total of about 2000 amyloid plaques were plaque sample but not sequenced in the control, it is still possible to find procured from four cortical sections in both cases. Non-plaque areas and quantify the peptide ion in MS survey scans of the control sample surrounding the plaques were captured as a control. Each CapSure according to its predicted m/z value and adjusted elution time. Other- Macro LCM cap (Arcturus, Mountain View, CA) was used for the wise, if the peptide could not be reliably located in the control sample, capture of about 500 plaques or control tissues from one slide. we estimated that the plaque/control ratio was more than 2-fold. Fi- Protein Extraction and Analysis by Mass Spectrometry—The caps nally, we accepted proteins that were found to be enriched at least were extracted twice with lysis buffer at 65 °C for 15 min. The lysis 2-fold in both AD cases. buffer was made from phosphate-buffered saline buffer, pH 7.2, with Immunohistochemistry—Nontransgenic and double transgenic the addition of 2% SDS, 10% glycerol, 10 mM dithiothreitol, 1 mM EDTA, C3/B6 mice expressing the APP695 isoform with the ”Swedish“ double and protease inhibitor mixture (Roche Applied Science). After the ex- mutation (APPswe) and PS1Ex9, a functional PS1 mutant lacking traction, the samples were alkylated with 50 mM iodoacetamide in the exon 9 (amino acids 290 –319) (36, 37), were used. Animals were anes- dark at room temperature for 30 min. The total amount of proteins in thetized with isoflurane (Abbott) and decapitated, and brains were the samples was estimated on a silver-stained SDS gel according to a removed and fixed in 4% paraformaldehyde for up to 6 h. Sections of standard protein marker with known concentration. cingulate cortex from four patients with autopsy-confirmed AD and two For mass spectrometry analysis, proteins in each sample were sep- normal controls were used. At autopsy, the brains were fixed with arated on a 6 –12% SDS gel (0.75 mm thick) and stained with Coomassie formalin for at least 48 h. Brain blocks were then sectioned (50 m) Blue G-250. The entire lane was cut into 15 pieces followed by in-gel with a freezing microtome (Microm, Heidelberg, Germany). The brain trypsin digestion (28). The resulting peptides from each gel piece were sections were cryoprotected in 40% sucrose in phosphate-buffered sa- dissolved in buffer A (0.4% acetic acid, 0.005% heptafluorobutyric acid, line buffer (50 mM phosphate-buffered saline, pH 7.2). Double labeling 5% acetonitrile). A pressure cell was used to load each sample onto a was performed by using rabbit anti-A (1:200, polyclonal antibody, 50-m inner diameter  12-cm self-packed, fused silica C18 capillary Chemicon, Temecula, CA) together with one of the following monoclonal column as described (29). Peptides were eluted during a 2-h gradient antibodies: anti-Hsp70/90 (1:200, Stressgen, Victoria, British Colum- from 10 to 30% buffer B (0.4% acetic acid, 0.005% heptafluorobutyric bia, Canada) or anti-vimentin (1:2000, Roche Applied Science). For A acid, 95% acetonitrile; flow rate, 300 nl/min). Eluted peptides were colocalization with rabbit anti-dynein heavy chain (1:200, Santa Cruz ionized under high voltage (1.8 –2 kV) and detected in an MS survey Biotechnology Inc., Santa Cruz, CA), a monoclonal antibody against A scan from 400 to 1700 atomic mass units with 2 microscans followed by (4G8, 1:200, Chemicon) was used. After primary antibody incubation three data-dependent MS/MS scans (3 microscans each, isolation width overnight at 4 °C, the sections were extensively rinsed and incubated 3 atomic mass units, 35% normalized collision energy, dynamic range 3 with fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody min) in a completely automated fashion on an LCQ-DECA XP-Plus ion (1:200, Jackson ImmunoResearch, Bar Harbor, ME) and then with trap mass spectrometer (Thermo Finnigan, San Jose, CA). biotinylated goat anti-mouse antibody (1:1000, Vector Laboratories, Proteomic Characterization of Amyloid Plaques 37063 FIG.2. Flow chart for the identification of proteins enriched in the plaque regions. Proteins identified in the plaques and the controls are represented by solid line circles and dashed line circles, FIG.1. Isolation of amyloid plaques by laser capture microdis- respectively. The number of overlapped proteins in both plaque samples section. A, the before and after images indicate the removal of a plaque is indicated in the relevant areas. A total of 256 proteins found in both region from a thioflavin-S-stained AD brain section. The isolated plaque AD plaques was processed in two successive steps: (i) removing proteins was attached to the cap as shown. Moreover, the surrounding non- that were found to be abundant in the non-plaque control and (ii) plaque regions were also captured as control on a different cap. B, quantifying the remaining 168 proteins based on extracted ion current protein in the captured plaques (p) and non-plaque control (c) were of corresponding peptides. Finally we found that 26 proteins were extracted by a SDS-containing lysis buffer. A small fraction (5%) of enriched at least 2-fold in the plaque regions, of which several proteins each sample and molecular weight marker (m) was run on an SDS gel were further verified by conventional immunohistochemistry. followed by silver staining as indicated. The remaining samples (95%, 4 g) were resolved on another SDS gel and stained with Coomassie Blue G-250 (data not shown). Each sample lane was cut into 15 pieces that were subjected to trypsin digestion and LC-MS/MS analysis. The cases, the protein components may differ in AD patients. We gel excision pattern is shown on the right according to the marker. repeated the entire proteomic analysis by using samples iso- lated from the second AD case and identified 413 proteins in Burlingame, CA) at room temperature for 2 h. The biotinylated second- the plaques and 384 proteins in its own non-plaque control ary antibody was further detected by avidin-biotin complex and Tyra- (Fig. 2). The data sets from the two AD cases were combined to mide Signal Amplification (PerkinElmer Life Sciences). The double- result in a list of 488 proteins detected in the plaque samples labeled sections were examined with a 40 objective lens (numeric (supplemental Table S1). The current literature identifies aperture 1.4) on a LSM510 laser-scanning confocal microscope (Zeiss). about 53 proteins present in the plaques, of which 44 proteins RESULTS were found in our large scale analyses (Table S1). As expected, Identification of Proteins Enriched in AD Amyloid Plaques by many of these proteins were also found in the cortical areas Quantitative Proteomics—Senile plaques in AD brain tissues without the plaques, because some normal cellular elements were stained with thioflavin-S and isolated by using LCM (Fig. are components of the plaques, including glia and neurons. 1A). Despite some background staining on the postmortem Alternatively, these proteins may reflect the capture of normal tissue sections, the plaques were easily distinguishable under cellular elements along with the plaques. the microscope. Approximately 2,000 plaques from one AD To determine which of the above proteins were enriched in brain were captured to yield 4 g of total protein after ex- the plaque regions, we utilized a strategy outlined in Fig. 2. traction with SDS-containing lysis buffer. The non-plaque re- First, only the 256 plaque proteins detected in both AD cases gions from the same brain sections were also procured as a were considered to increase the dataset reliability. Although it control. The total protein samples extracted from the plaques is difficult to estimate protein abundance directly from its and the non-plaque control were resolved in parallel on an SDS peptide ion current, the number of peptides identified for each gel (Fig. 1B), which indicates a similar protein composition in protein is roughly correlated with the abundance of the protein the two samples. The proteins in the entire gel lanes were after the protein size is normalized (38, 39). Based on this analyzed by LC-MS/MS as described under “Materials and principle, we removed the proteins that were identified more Methods.” Approximately 30,000 MS/MS spectra were acquired frequently in the controls than in the plaques according to the for each sample and searched against a human protein data- corresponding peptide numbers (Fig. 2 and Table S1), and we base by using the Sequest program (30). The matched peptides kept the remaining 168 proteins for further quantitative anal- were further filtered rigorously and led to the identification of ysis. More recently, several groups (15, 34, 35) proposed a 331 proteins in the plaques and 327 proteins in the adjacent simple relative quantification method via extracted ion current non-plaque regions of the cortex in this AD case (Fig. 2). of peptides in successive analyses. We used this method to As the plaques are complex and heterogeneous neuropatho- quantify the 168 proteins by manual inspection of the raw files. logical structures and are expected to vary among human For example, during the LC-MS/MS analysis, an A tryptic 37064 Proteomic Characterization of Amyloid Plaques FIG.3. Identification and quantification of A by reverse-phase LC-MS/MS. A, comparison of MS survey scans shows that a peptide ion with m/z 663.8 is much more abundant in the plaques (lower panel) than in the non-plaque control (upper panel). A partial region (620 –700 m/z) is shown for simplicity, although the entire scan range is from 400 to 1700 m/z. B, MS/MS scan of the precursor ion (663.8 m/z), which led to the identification of a tryptic peptide derived from A. Gaps between the adjacent product ions fit the mass of amino acid residues as indicated. C, the elution profile of the A tryptic peptide (m/z 663.4 – 666.4 due to naturally occurring isotopic distribution) from the non-plaque control and the plaque sample after normalizing the elution time. The peptide was quantified based on the extracted ion current. In fact, the data acquired in LC-MS/MS are three-dimensional (ion current indicated by the relative abundance, retention time of LC, and the m/z measurement every 2 s during the entire LC). A was a snapshot of m/z measurement at the retention time point of 65 min in C. peptide was detected in an MS survey scan (Fig. 3A) and ples, we grouped them under functional categories in alphabet- sequenced by MS/MS (Fig. 3B) in the plaque sample and the ical order (Table I and Table S1) and discuss the implication of control. The extracted ion current signal for the peptide is some enriched plaque proteins in AD pathogenesis. shown in Fig. 3C, which allows for peptide quantification in Cell Adhesion and Cytoskeleton and Membrane Traffick- both samples. It is worth noting that a trypsin auto-cleavage ing—A number of intercellular adhesion molecules have been peptide was used as an internal standard to normalize the shown to be localized in amyloid plaques in AD patients (40). experimental variation and to fit the elution time of selected We found that collagen I and fibrinogen were concentrated in peptides. The plaque/control ratio of A abundance was deter- the plaques. In addition to extracellular structures, integrity of mined to be around 80, indicative of the enormous enrichment the intracellular cytoskeleton is important for neuronal physi- of A in the plaques. The relative abundance of all other pro- ological functions such as axoplasmic flow of essential synaptic teins was quantified in a similar manner, resulting in the final components (41). In our study, all major isoforms of cytoskel- acceptance of 26 proteins enriched at least 2-fold in the plaques etal components, actin, tubulin, and neurofilament, were iden- of both AD cases (Table I). However, the majority of previously tified with high numbers of peptides, which indicates that they identified plaque proteins is not on this list because some are abundant species in the plaques as well as in non-plaque abundant proteins (e.g. actin and tubulin, Table S1) are present regions. Most interestingly, we identified one actin-binding but not necessarily concentrated in the plaque, and some low protein, coronin, concentrated in the plaque, although its po- abundance proteins (e.g. collagen XXV and antichymotrysin, tential role in pathogenesis remains unclear. Also enriched in Table S1) were detected only in one AD case perhaps due to plaques is the microtubule-associated protein tau that is more sensitivity limitation of the LC-MS/MS approach. Neverthe- commonly known to be associated with neurofibrillary tangles less, the list of 26 proteins that were identified and concen- and neurites (42) and likely represents the dystrophic neurite trated in the plaque regions in both AD cases are more likely to component in the plaques. The identification of a variety of be genuine plaque proteins. cytoskeletal protein elements, some of which are known to be Classification of Identified Plaque Proteins—To evaluate the relevant to AD, suggests that these elements may be involved proteins identified in the plaques from AD postmortem sam- in plaque formation, and cytoskeletal impairments may lead to Proteomic Characterization of Amyloid Plaques 37065 TABLE I List of 26 proteins enriched in amyloid plaques as compared with non-plaque areas The proteins and functional categories were sorted in the alphabetic order. When a peptide in the non-plaque control was not detected, we assumed the ratio was more than 2. Sometimes, even if a peptide ion was not sequenced by MS/MS, it was still possible to identify it in MS survey scan to enable quantification (see “Materials and Methods”). AD case 1 AD case 2 TM Protein name GenBank accession no. a b c Ctl Plaque P/C Ctl Plaque P/C Cell adhesion Collagen I, -1 polypeptide NP_000079.1 0 2 3.6 0 1 2 Fibrinogen,  NP_000500.1 0 3 20 2 2 Channels/receptors ATPase, Ca -transporting NP_001674.1 1 3 2.1 0 2 2 Chaperones Heat shock 90-kDa protein 1,  NP_031381.2 7 9 2.1 11 14 2.0 Cytoskeleton Coronin, actin-binding protein NP_009005.1 0 1 20 1 2 tau NP_058519.1 1 3 6.8 2 4 3.2 Inflammation Glial fibrillary acidic protein (GFAP) NP_002046.1 16 21 3.6 20 24 2.1 Vimentin NP_003371.1 4 12 9 11 15 2.3 Kinases/phosphatases and regulators 14-3-3 / NP_003395.1 2 4 3.1 5 8 3.5 14-3-3  NP_006752.1 2 5 6.1 7 9 4.4 14-3-3  NP_003397.1 4 6 7.7 5 10 7.4 Membrane trafficking Clathrin, heavy polypeptide 1 NP_001826.1 0 1 2 0 5 2.7 Dynamin 1 NP_004399.1 5 6 2.3 18 23 2.2 Dynein, heavy polypeptide 1 NP_001367.2 0 1 4.8 0 1 2 Metabolism Phosphofructokinase NP_002618.1 0 2 3.9 3 7 2.5 Others Amyloid -peptide NP_000475.1 1 1 80.0 0 1 2 Proteolysis Antitrypsin NP_000286.2 0 3 2 1 2 2.5 ATPase, H -transporting, lysosomal V0 subunit A NP_005168.2 0 1 2 1 3 2.4 ATPase, H -transporting, lysosomal V1 subunit B NP_001684.2 0 4 2.1 4 8 3.0 ATPase, H -transporting, lysosomal V1 subunit D NP_057078.1 0 1 2 1 3 2.6 ATPase, H -transporting, lysosomal V1 subunit E NP_001687.1 1 2 5.0 4 5 8.3 Cathepsin D NP_001900.1 0 1 20 3 2 Cystatin B NP_000091.1 1 2 2.9 0 1 2 Cystatin C NP_000090.1 0 1 20 1 2 Ubiquitin-activating enzyme E1 NP_695012.1 0 1 7.1 3 4 2.0 Vacuolar ATPase subunit H NP_057025.1 0 1 2 1 3 2.1 Ctl indicates the number of different peptides that identify a protein in the control samples of non-plaque regions. Plaque indicates the number of peptides that identify a protein in the plaque samples. P/C indicates the abundance ratio of proteins from the plaque samples versus non-plaque regions. a deficit in axoplasmic flow and eventually to neuritic dystro- ent in neurofibrillary tangles (46), and at least one 14-3-3 phy. Indeed, numerous proteins involved in membrane traffick- protein has further been shown to be an effector of tau phos- ing and protein sorting were revealed to be concentrated in the phorylation (47). Moreover, we attempted to detect protein plaques by the mass spectrometry analysis, such as clathrin phosphorylation sites in the plaque samples by tandem mass heavy chain, dynamin, and dynein heavy chain (Table I). This spectrometry, and we located two phosphorylated amino acid observation implicates that the AD plaque might sequester residues in neurofilament 3 (SPVPKSPVEEK and KAESPV- some key proteins to perturb the protein sorting system that is KEEAVAEVVTITK with modified sites shown in boldface with crucial for maintaining normal synaptic plasticity. underline). The first phosphorylation site was documented in Chaperones and Inflammation—It is well known that the tandem repeats in the neurofilament sequence and was exces- senile plaque core is surrounded by activated astrocytes, mi- sively modified in the AD brain (48). The second peptide indi- croglia, and dystrophic neurites (43, 44), and the heat shock cated a phosphorylation site on serine 736 that we show for the proteins exhibit high expression levels in reactive astrocytes in first time. These phosphorylation events may play a role in the areas rich in senile plaques (45). Consistently, we identified formation of dystrophic neurites surrounding the plaque core. many heat shock proteins (Table S1) and found that HSP90 Proteolysis—The ubiquitin-proteasome system plays a cru- was enriched in the plaques (Table I). We also identified glial cial role in the degradation of misfolded proteins and turnover fibrillary acidic protein and vimentin with high numbers of of cell signaling molecules (49). This study identified the ubiq- peptides; both are the major components of intermediate fila- uitin-activating enzyme enriched in the plaques. More strik- ments in activated glial cells. ingly, numerous subunits of lysosomal ATPase and cathepsin D Kinase/Phosphatase and Regulators—The imbalance of were found to be concentrated in the plaques, suggesting the phosphorylation/dephosphorylation activity is believed to con- high proteolytic activity in the plaques versus non-plaque re- tribute to AD pathology, as evidenced by tau hyperphosphory- gions. Moreover, we found in the plaques antitrypsin, cystatin lation in the neurofibrillary tangles. We identified multiple B, and cystatin C. Cystatin C is a cysteine proteinase inhibitor kinases (Table S1), but none of them were specifically enriched and has been shown to be up-regulated in AD and coaggregated in the plaque regions. Instead, three 14-3-3 isoforms showed a with A (50, 51). Cystatin C is also proposed as a potential risk significant degree of enrichment in the isolated plaques. Pre- factor for late-onset AD (52). Overall, the relative abundance of vious studies have demonstrated that 14-3-3 proteins are pres- proteolytic enzymes and inhibitors in multiple cellular proteo- 37066 Proteomic Characterization of Amyloid Plaques mice manifested high density of amyloid plaques, the majority of which is clearly surrounded by dynein immunoreactive structures that may represent enlarged neurites (Fig. 4F). Re- cently, missense mutations in dynein heavy chain have been genetically linked to progressive motor neuron degeneration and the formation of Lewy body-like inclusions (55). It has also been observed previously (56) that dynein immunoreactivity in AD brain tissue is significantly increased compared with nor- mal brain. Given that dynein is responsible for retrograde transport of vesicles along microtubules in the axon (57), the enrichment of dynein encircling the plaque core might trigger trafficking malfunction to cause neuritic dystrophy. DISCUSSION Our studies revealed a total of 488 proteins in amyloid plaques, representing three histopathological components: the plaque core, activated glial cells, and dystrophic neurites. Iden- tified intracellular proteins are likely derived from neurites and/or glial cells surrounding the plaque core, whereas extra- cellular proteins may be components of the plaque core. It is also possible that some intracellular proteins can leak out after plasma membrane damage during neuronal degeneration. By quantitative mass spectrometry, we further identified 26 pro- teins enriched in the plaques when comparing with the non- plaque control sample from the same AD case. This comparison is particularly valuable because both samples were derived from the same postmortem tissue specimen, which essentially eliminates the effects of many confounding factors (e.g. genetic variation, postmortem interval, etc.) that are often encountered FIG.4. Localization of plaque proteins by immunofluores- in human tissue studies. cence confocal microscopy. Sections of human postmortem AD neo- Our results confirmed the presence of most proteins that cortex were stained with A antibody (either polyclonal anti-A , A were detected previously in the plaques mainly by immunohis- and B, or monoclonal antibody 4G8, C and D, green) and antibodies tochemistry (40). The classic plaque components identified in against vimentin (A, red), Hsp70/90 complex (B, red), or dynein heavy this study include A, 1-antichymotrypsin (58), apolipopro- chain (C and D, red). Four serial confocal slices through a plaque region are shown in D ata1-m optical slice thickness and 4-m intervals tein E (59), collagen type XXV (60), cystatin C (61), -synuclein between slices. Brain sections containing cortex and hippocampus from (62), proteoglycans (63), and clusterin (64). On the other hand, nontransgenic (E) and PS1/APP double transgenic mice (F) at 9 months only a few proteins known to be amyloid plaque components of age were labeled with monoclonal A antibody 4G8 (green) and were missed in this study. Those included complement inhibi- polyclonal heavy chain antibodies (red). Scale bar, A, B, D, and the insert in F,50 m; C, E, and F, 100 m. tors (40), myeloperoxidase (5),  -macroglobin (65), superoxide dismutase (66), HO-1 (67), catalase (68), and cholinesterase lytic pathways strongly suggests the activation of protein deg- (69). The apparent absence of these proteins in the plaques in radation mechanisms and the interplay between proteolysis our study could be due to the low abundance of the proteins and inhibition activities during the plaque formation. and/or incompatibility of tryptic peptides with the LC-MS/MS Validation of Selected Plaque Components—To verify further system (12). Our results from two independent studies of AD the localization of some of the proteins identified, we undertook cases revealed that about two-thirds of the proteins were iden- immunostaining analysis on postmortem brain tissues for their tified in both cases. The difference between the two data sets colocalization with A. In Fig. 4A, monoclonal antibody against may be contributed by the sample variation in the patients and vimentin specifically labeled activated astrocytes surrounding the nature of shotgun proteomics strategy, as only a fraction of the fibrillary plaque core that was recognized by A antibody, peptides was selected and sequenced by mass spectrometry and some astrocyte processes were extended deeply into the when a complex peptide mixture was analyzed by the LC- plaque core, indicating a local inflammatory response in the MS/MS approach (29). plaque region (53, 54). Polyclonal antibodies against the Hsp70 Immunohistochemistry was employed as an independent ap- and Hsp90 complex show strong immunoreactive signals in proach to confirm the proteomic findings. Eight proteins (in dot-like structure located in the plaque regions (Fig. 4B), con- Table I and Table S1) were selected largely based on the avail- sistent with our proteomic analysis. More strikingly, dynein ability of the antibodies. Most of them are concentrated by heavy chain was detected as punctate and thread-like struc- varying degrees in the plaque regions, as exemplified by vimen- tures in many plaques labeled by A antibody (Fig. 4C), sug- tin, Hsp 70/90 complex, dynein (Fig. 4), -synuclein, glial fibril- gesting that dynein heavy chain is a constituent of plaque lary acidic protein, and synaptophysin (data not shown). Other neurites. Confocal microscopic images clearly indicated that proteins like actin and tubulin display a broad distribution but dynein heavy chain was enriched in the plaque, compared with are not specifically concentrated in the plaques (data not the surrounding neuropil (Fig. 4D). shown), although actin and tubulin are believed to be plaque The colocalization of dynein with amyloid plaque was corrob- components. Therefore, the list of 26 proteins according to orated in an AD model, PS1/APP double transgenic mice (36, quantitative analysis should contain much less false plaque 37). As expected, no plaque was visualized in 9-month-old constituents. control animals, and dynein heavy chain antibodies labeled the A is widely accepted as the major component of the amyloid soma and neurites in the cortex and hippocampus (Fig. 4E). In plaque core; consistently, our mass spectrometry analysis iden- great contrast, the similar brain regions in 9-month-old AD tified A in both plaque and non-plaque control but showed it Proteomic Characterization of Amyloid Plaques 37067 to be enriched about 80-fold in the plaques. No other proteins the extracted ion current of eluted peptides. More accurate were found to accrue at a similar level, suggesting that A is quantification of proteins could be achieved by applying stable the sole major protein species in the plaque core. Although A isotope labeling based techniques such as isotope-coded affinity aggregates are resistant to extraction by many reagents except tag strategy (73). To our knowledge, this is the first large scale analysis of for 70% formic acid (7), in order to extract other coaggregated proteins of interest, we chose 2% SDS among several tested proteins from AD amyloid plaques. The results of this study demonstrate that the protein molecules in amyloid plaques are extraction conditions because this buffer led to the best yield of highly complex and diverse, implicating the involvement of total proteins. As is well known, SDS dissolves A fibrils poorly many cellular pathways in disease development. The plaque (7), and accordingly, we identified only weak A immunoreac- subproteome identified in our study will be instructive for tivity in the SDS-solubilized plaque sample but not in the subsequent hypothesis-driven experiments on disease biomar- non-plaque control by Western blotting (data not shown). How- ker identification and molecular genesis of Alzheimer’s disease. ever, the small amount of A warranted strong signals in the LC-MS/MS analysis, which strongly suggested that many other Acknowledgments—We thank Dr. Michael Iuvone and James Wessel major protein aggregates in the plaques could barely elude for their help in LCM. We also thank Dr. Steven Gygi, Carson Thoreen, detection. and Rob Duarte for their help in computational analysis; Stephanie Carter for maintaining the animals; Sara Dodson for making mouse One major concern with this type of analysis is the purity of brain sections. In addition, we are grateful to Drs. Victor Faundez, the plaque sample, because LC-MS/MS can detect protein spe- John Wood, and Deanna Smith for providing antibodies. cies with high sensitivity. We used the LCM approach for the isolation of amyloid plaques from postmortem tissues stained REFERENCES by thioflavin-S. This dye preferentially labels proteins with 1. Hardy, J., and Selkoe, D. J. 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Proteomic Characterization of Postmortem Amyloid Plaques Isolated by Laser Capture Microdissection

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 35, Issue of August 27, pp. 37061–37068, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Proteomic Characterization of Postmortem Amyloid Plaques □ S Isolated by Laser Capture Microdissection* Received for publication, April 2, 2004, and in revised form, June 24, 2004 Published, JBC Papers in Press, June 25, 2004, DOI 10.1074/jbc.M403672200 Lujian Liao‡§, Dongmei Cheng‡§, Jian Wang‡§, Duc M. Duong‡§, Tatyana G. Losik‡§, Marla Gearing§ , Howard D. Rees§ , James J. Lah§ , Allan I. Levey§ , and Junmin Peng‡§** From the ‡Department of Human Genetics, §Center for Neurodegenerative Disease, the Department of Pathology and Laboratory Medicine, and the Department of Neurology, Emory University School of Medicine, Atlanta, Georgia 30322 The first major breakthrough in understanding the molecu- The presence of amyloid plaques in the brain is one of the pathological hallmarks of Alzheimer’s disease (AD). lar pathogenesis of AD came from the biochemical purification We report here a comprehensive proteomic analysis of of amyloid -peptide (A) from senile plaques, as described by senile plaques from postmortem AD brain tissues. Senile Glenner and Wong (6), and the subsequent sequencing and plaques labeled with thioflavin-S were procured by la- identification of the A precursor protein (APP) gene. Although ser capture microdissection, and their protein compo- the major insoluble component of plaques has been identified nents were analyzed by liquid chromatography coupled as A (6, 7), the entire molecular composition of the plaques is with tandem mass spectrometry. We identified a total of not known. The plaques are highly complex structures with a 488 proteins coisolated with the plaques, and we found variety of neural and glial elements (8), and many proteins multiple phosphorylation sites on the neurofilament in- have been localized to these structures by immunohistochem- termediate chain, implicating the complexity and diver- istry. However, biochemical verification of the plaque compo- sity of cellular processes involved in the plaque forma- nents has been scarce. Moreover, biochemical approaches pre- tion. More significantly, we identified 26 proteins viously applied to purify plaque components generally use very enriched in the plaques of two AD cases by quantitative stringent extraction conditions (e.g. high concentration of salt, comparison with surrounding non-plaque tissues. The urea, and/or protease treatment) that may remove A-associ- localization of several proteins in the plaques was fur- ther confirmed by the approach of immunohistochemis- ated proteins. The identities and roles of other plaque proteins try. In addition to previously identified plaque constit- that may act synergistically or competitively with A in aggre- uents, we discovered novel association of dynein heavy gation and deposition are also incomplete. A systematic anal- chain with the plaques in human postmortem brain and ysis of plaque proteins should shed light on the underlying in a double transgenic AD mouse model, suggesting that molecular processes that govern the plaque formation. neuronal transport may play a role in neuritic degener- Traditionally, proteomic analysis is performed by comparing ation. Overall, our results revealed for the first time the samples between AD cases and normal controls in two-dimen- sub-proteome of amyloid plaques that is important for sional gels and identifying proteins of interest by mass spec- further studies on disease biomarker identification and trometry (9). Several groups (10, 11) have tried this strategy molecular mechanisms of AD pathogenesis. and identified some proteins that are altered in the expression levels. However, the two-dimensional gel method is generally 1 incompatible with proteins of extreme size, pI, and/or hydro- Alzheimer’s disease (AD) is a devastating neurological dis- phobicity, and it is tedious to determine the identity of hun- order that impairs cognitive function and disturbs emotion and dreds of protein spots displayed on a two-dimensional gel. personality. Histopathologically, AD is manifested by the ex- Many of the limitations can be overcome by the development of tracellular aggregation of amyloid plaques and the intraneuro- liquid chromatography combined with tandem mass spectrom- nal neurofibrillary tangles. Although current amyloid cascade etry (LC-MS/MS) (12, 13). It is now possible to analyze hun- hypothesis (1) or tau hypothesis (2) provides a framework for dreds to thousands of proteins directly from a complex protein studying AD pathogenesis, the detailed molecular mechanisms mixture, and the sensitivity can reach low femtomole and even that translate amyloid or tau accumulation into neuronal dam- subfemtomole levels. The power of shotgun LC-MS/MS-based age and functional brain impairments are largely unknown. In proteomics technology has been documented by successful pro- addition, there are numerous, complex pathological changes in teomic analysis of subcellular structures in mammalian cells, AD brain that contribute to neuronal and synaptic degenera- such as nucleolus (14), centrosome (15), nuclear envelope (16), tion, including mitochondrial dysfunction, oxidative damage, and inflammation (3–5). mitochondria (17), and postsynaptic density (18). The method- ology has also been used to address dynamic changes in protein levels under specific conditions, as exemplified by the identifi- * The costs of publication of this article were defrayed in part by the cation of protein components in the epidermal growth factor payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to signaling pathway (19) and the functional investigation of the indicate this fact. myc oncogene (20). Mass spectrometry has also greatly simpli- □ S The on-line version of this article (available at http://www.jbc.org) fied the analysis of post-translational modifications (21, 22). contains Table S1. ** To whom correspondence should be addressed. E-mail: jpeng@ Whereas it may be problematic to isolate plaques to homoge- genetics.emory.edu. neity by using traditional biochemical methods, the advent of The abbreviations used are: AD, Alzheimer’s disease; LC-MS/MS, laser capture microdissection (LCM) allows procuring a micro- liquid chromatography coupled with tandem mass spectrometry; LCM, scopic region as small as 3–5 m in diameter (23). The LCM laser capture microdissection; A, amyloid -peptide; APP, A precur- sor protein. technology has been used extensively to collect homogeneous cell This paper is available on line at http://www.jbc.org 37061 This is an Open Access article under the CC BY license. 37062 Proteomic Characterization of Amyloid Plaques Database Searching for Protein Identification—The Sequest algo- types for DNA mutation detection and gene expression analysis rithm (30) was utilized for searching all MS/MS spectra against the at the mRNA level (23). More recently, its application in proteom- human reference data base (ftp.ncbi.nih.gov/genbank, July, 2003). The ics has begun to be appreciated (24, 25). It is possible to use LCM parameters were set to allow parent ion mass tolerance to be three and to capture neuropathological structures with high purity, but the to consider only the b and y ion series. Modifications were permitted to amount of samples collected by LCM is often limited, complicat- allow the detection of the following (mass shift shown in daltons): ing proteomic analysis by two-dimensional gel-based methods. oxidized methionine (16), carboxymethylated cysteine (57), and phosphorylated serine, threonine, and tyrosine (80). We used more These limitations can be alleviated by the integration of LCM stringent Sequest criteria than described previously (31, 32): 1) only with the highly sensitive LC-MS/MS technology. fully tryptic peptides were considered; 2) Cn score is at least 0.08; and Here we present the isolation of postmortem amyloid plaques 3) Xcorr should be larger than 2.0, 1.7, or 3.3 for charge states of 1, 2, labeled with thioflavin-S by using the LCM approach and the 3, respectively. To reduce false-positives further, we manually veri- systematic identification of extracted proteins by LC-MS/MS. fied proteins matched by less than three peptides, because no false- positives were found among proteins identified by at least three distinct The entire procedure was performed twice with samples from two peptides (32). Therefore, all peptides were accepted with high confi- AD cases. Together, more than 480 proteins were detected in the dence. The conversion from the identified peptides to proteins was plaque samples, and 26 proteins were shown to be enriched in the complicated by the presence of different names for the same protein plaques in comparison with the surrounding tissue. The presence and/or by the sharing of peptides within several proteins (e.g. protein of selected proteins in the plaques was confirmed by immunohis- paralogs) (33). Thus we manually verified all proteins and removed the redundancy. Typically, we accepted proteins identified by at least one tochemistry, and cytoplasmic dynein was found to be a novel “unique peptide.” Obvious contaminants such as trypsin and keratins component of amyloid plaques in human specimens and in an AD were removed. Finally we merged the data sets of the plaque samples mouse model. These studies demonstrate a powerful new ap- from two independent experiments. proach for achieving comprehensive analysis of the sub-proteome Protein Quantification by Mass Spectrometry—Quantitative protein of neuropathological structures. comparison between the plaques and the non-plaque control was car- ried out in two steps. The first step was based on the number of peptides MATERIALS AND METHODS identified for an assigned protein, indicative of protein abundance. We discarded proteins that were identified by more peptides in the control Brain Sections—Small blocks of fresh frontal and temporal cortex than in the plaques from either AD case. The second step was based on were obtained at autopsy from a 55-year-old Caucasian female (post- extracted ion current of corresponding peptides in MS survey scans (15, mortem interval, 4.5 h, case 1) and a 78-year-old Caucasian male 34, 35). The ratio of peak intensities of selected peptides was measured (postmortem interval, 17.5 h, case 2). Both cases were neuropathologi- in the 15 pairs of peptide mixtures that were generated by in-gel cally diagnosed as definite AD according to CERAD criteria (26). The digestion of the 15 pairs of gel pieces as shown (Fig. 1). We analyzed brain blocks were embedded in Tissue-Tek® OCT Compound (Jed Pella each pair of samples in two consecutive LC-MS/MS runs on the same Inc., Redding, CA), frozen on dry ice, and stored at 80 °C. 10-m-thick column and found that the quantitative variation was within 2-fold in brain sections were cut by using a Leica CM 3050 cryostat (Leica general by using a trypsin auto-cleavage peptide (VATVSLPR, m/z Microsystems Inc., Bannockburn, IL) and mounted on uncoated and 842.5 for singly charged ion) as internal control. Therefore, we used uncharged glass slides. 2-fold as the threshold for protein enrichment in the plaques. The Laser Capture Microdissection—Isolation of plaques by LCM was trypsin auto-cleavage peptide was also used to normalize the measured performed based on the protocol developed previously (27). The frozen peptide ratios and to normalize the elution time of selected peptides brain sections were thawed at room temperature, fixed with 75% eth- between the pair of LC-MS/MS analyses. It should be mentioned that, anol for 1 min, stained with 1% thioflavin-S for 1 min, differentiated in in contrast to the peptide identification that was primarily derived from 75% ethanol for 1 min, and then subjected to dehydration in a series of its MS/MS spectrum (Fig. 3B), the peptide quantification relied on the graded ethanol. Finally, the slides were cleared in xylene for 5 min and MS survey scan (Fig. 3A). An MS survey scan allowed the detection of then air-dried and desiccated. LCM was performed the same day under many peptide ion peaks, of which only the most three predominant were a fluorescence microscope attached to a Pixcell II laser capture facility selected for sequencing by MS/MS analysis. However, the other nonse- (Arcturus, Mountain View, CA) with the following settings: excitation quenced peaks on MS survey scans could be useful for quantification. wavelength, 495 nm; laser power, 60 – 80 milliwatts; duration, 1 ms; For example, when a peptide was sequenced by MS/MS analysis in the and laser spot size, 7.5 m. A total of about 2000 amyloid plaques were plaque sample but not sequenced in the control, it is still possible to find procured from four cortical sections in both cases. Non-plaque areas and quantify the peptide ion in MS survey scans of the control sample surrounding the plaques were captured as a control. Each CapSure according to its predicted m/z value and adjusted elution time. Other- Macro LCM cap (Arcturus, Mountain View, CA) was used for the wise, if the peptide could not be reliably located in the control sample, capture of about 500 plaques or control tissues from one slide. we estimated that the plaque/control ratio was more than 2-fold. Fi- Protein Extraction and Analysis by Mass Spectrometry—The caps nally, we accepted proteins that were found to be enriched at least were extracted twice with lysis buffer at 65 °C for 15 min. The lysis 2-fold in both AD cases. buffer was made from phosphate-buffered saline buffer, pH 7.2, with Immunohistochemistry—Nontransgenic and double transgenic the addition of 2% SDS, 10% glycerol, 10 mM dithiothreitol, 1 mM EDTA, C3/B6 mice expressing the APP695 isoform with the ”Swedish“ double and protease inhibitor mixture (Roche Applied Science). After the ex- mutation (APPswe) and PS1Ex9, a functional PS1 mutant lacking traction, the samples were alkylated with 50 mM iodoacetamide in the exon 9 (amino acids 290 –319) (36, 37), were used. Animals were anes- dark at room temperature for 30 min. The total amount of proteins in thetized with isoflurane (Abbott) and decapitated, and brains were the samples was estimated on a silver-stained SDS gel according to a removed and fixed in 4% paraformaldehyde for up to 6 h. Sections of standard protein marker with known concentration. cingulate cortex from four patients with autopsy-confirmed AD and two For mass spectrometry analysis, proteins in each sample were sep- normal controls were used. At autopsy, the brains were fixed with arated on a 6 –12% SDS gel (0.75 mm thick) and stained with Coomassie formalin for at least 48 h. Brain blocks were then sectioned (50 m) Blue G-250. The entire lane was cut into 15 pieces followed by in-gel with a freezing microtome (Microm, Heidelberg, Germany). The brain trypsin digestion (28). The resulting peptides from each gel piece were sections were cryoprotected in 40% sucrose in phosphate-buffered sa- dissolved in buffer A (0.4% acetic acid, 0.005% heptafluorobutyric acid, line buffer (50 mM phosphate-buffered saline, pH 7.2). Double labeling 5% acetonitrile). A pressure cell was used to load each sample onto a was performed by using rabbit anti-A (1:200, polyclonal antibody, 50-m inner diameter  12-cm self-packed, fused silica C18 capillary Chemicon, Temecula, CA) together with one of the following monoclonal column as described (29). Peptides were eluted during a 2-h gradient antibodies: anti-Hsp70/90 (1:200, Stressgen, Victoria, British Colum- from 10 to 30% buffer B (0.4% acetic acid, 0.005% heptafluorobutyric bia, Canada) or anti-vimentin (1:2000, Roche Applied Science). For A acid, 95% acetonitrile; flow rate, 300 nl/min). Eluted peptides were colocalization with rabbit anti-dynein heavy chain (1:200, Santa Cruz ionized under high voltage (1.8 –2 kV) and detected in an MS survey Biotechnology Inc., Santa Cruz, CA), a monoclonal antibody against A scan from 400 to 1700 atomic mass units with 2 microscans followed by (4G8, 1:200, Chemicon) was used. After primary antibody incubation three data-dependent MS/MS scans (3 microscans each, isolation width overnight at 4 °C, the sections were extensively rinsed and incubated 3 atomic mass units, 35% normalized collision energy, dynamic range 3 with fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody min) in a completely automated fashion on an LCQ-DECA XP-Plus ion (1:200, Jackson ImmunoResearch, Bar Harbor, ME) and then with trap mass spectrometer (Thermo Finnigan, San Jose, CA). biotinylated goat anti-mouse antibody (1:1000, Vector Laboratories, Proteomic Characterization of Amyloid Plaques 37063 FIG.2. Flow chart for the identification of proteins enriched in the plaque regions. Proteins identified in the plaques and the controls are represented by solid line circles and dashed line circles, FIG.1. Isolation of amyloid plaques by laser capture microdis- respectively. The number of overlapped proteins in both plaque samples section. A, the before and after images indicate the removal of a plaque is indicated in the relevant areas. A total of 256 proteins found in both region from a thioflavin-S-stained AD brain section. The isolated plaque AD plaques was processed in two successive steps: (i) removing proteins was attached to the cap as shown. Moreover, the surrounding non- that were found to be abundant in the non-plaque control and (ii) plaque regions were also captured as control on a different cap. B, quantifying the remaining 168 proteins based on extracted ion current protein in the captured plaques (p) and non-plaque control (c) were of corresponding peptides. Finally we found that 26 proteins were extracted by a SDS-containing lysis buffer. A small fraction (5%) of enriched at least 2-fold in the plaque regions, of which several proteins each sample and molecular weight marker (m) was run on an SDS gel were further verified by conventional immunohistochemistry. followed by silver staining as indicated. The remaining samples (95%, 4 g) were resolved on another SDS gel and stained with Coomassie Blue G-250 (data not shown). Each sample lane was cut into 15 pieces that were subjected to trypsin digestion and LC-MS/MS analysis. The cases, the protein components may differ in AD patients. We gel excision pattern is shown on the right according to the marker. repeated the entire proteomic analysis by using samples iso- lated from the second AD case and identified 413 proteins in Burlingame, CA) at room temperature for 2 h. The biotinylated second- the plaques and 384 proteins in its own non-plaque control ary antibody was further detected by avidin-biotin complex and Tyra- (Fig. 2). The data sets from the two AD cases were combined to mide Signal Amplification (PerkinElmer Life Sciences). The double- result in a list of 488 proteins detected in the plaque samples labeled sections were examined with a 40 objective lens (numeric (supplemental Table S1). The current literature identifies aperture 1.4) on a LSM510 laser-scanning confocal microscope (Zeiss). about 53 proteins present in the plaques, of which 44 proteins RESULTS were found in our large scale analyses (Table S1). As expected, Identification of Proteins Enriched in AD Amyloid Plaques by many of these proteins were also found in the cortical areas Quantitative Proteomics—Senile plaques in AD brain tissues without the plaques, because some normal cellular elements were stained with thioflavin-S and isolated by using LCM (Fig. are components of the plaques, including glia and neurons. 1A). Despite some background staining on the postmortem Alternatively, these proteins may reflect the capture of normal tissue sections, the plaques were easily distinguishable under cellular elements along with the plaques. the microscope. Approximately 2,000 plaques from one AD To determine which of the above proteins were enriched in brain were captured to yield 4 g of total protein after ex- the plaque regions, we utilized a strategy outlined in Fig. 2. traction with SDS-containing lysis buffer. The non-plaque re- First, only the 256 plaque proteins detected in both AD cases gions from the same brain sections were also procured as a were considered to increase the dataset reliability. Although it control. The total protein samples extracted from the plaques is difficult to estimate protein abundance directly from its and the non-plaque control were resolved in parallel on an SDS peptide ion current, the number of peptides identified for each gel (Fig. 1B), which indicates a similar protein composition in protein is roughly correlated with the abundance of the protein the two samples. The proteins in the entire gel lanes were after the protein size is normalized (38, 39). Based on this analyzed by LC-MS/MS as described under “Materials and principle, we removed the proteins that were identified more Methods.” Approximately 30,000 MS/MS spectra were acquired frequently in the controls than in the plaques according to the for each sample and searched against a human protein data- corresponding peptide numbers (Fig. 2 and Table S1), and we base by using the Sequest program (30). The matched peptides kept the remaining 168 proteins for further quantitative anal- were further filtered rigorously and led to the identification of ysis. More recently, several groups (15, 34, 35) proposed a 331 proteins in the plaques and 327 proteins in the adjacent simple relative quantification method via extracted ion current non-plaque regions of the cortex in this AD case (Fig. 2). of peptides in successive analyses. We used this method to As the plaques are complex and heterogeneous neuropatho- quantify the 168 proteins by manual inspection of the raw files. logical structures and are expected to vary among human For example, during the LC-MS/MS analysis, an A tryptic 37064 Proteomic Characterization of Amyloid Plaques FIG.3. Identification and quantification of A by reverse-phase LC-MS/MS. A, comparison of MS survey scans shows that a peptide ion with m/z 663.8 is much more abundant in the plaques (lower panel) than in the non-plaque control (upper panel). A partial region (620 –700 m/z) is shown for simplicity, although the entire scan range is from 400 to 1700 m/z. B, MS/MS scan of the precursor ion (663.8 m/z), which led to the identification of a tryptic peptide derived from A. Gaps between the adjacent product ions fit the mass of amino acid residues as indicated. C, the elution profile of the A tryptic peptide (m/z 663.4 – 666.4 due to naturally occurring isotopic distribution) from the non-plaque control and the plaque sample after normalizing the elution time. The peptide was quantified based on the extracted ion current. In fact, the data acquired in LC-MS/MS are three-dimensional (ion current indicated by the relative abundance, retention time of LC, and the m/z measurement every 2 s during the entire LC). A was a snapshot of m/z measurement at the retention time point of 65 min in C. peptide was detected in an MS survey scan (Fig. 3A) and ples, we grouped them under functional categories in alphabet- sequenced by MS/MS (Fig. 3B) in the plaque sample and the ical order (Table I and Table S1) and discuss the implication of control. The extracted ion current signal for the peptide is some enriched plaque proteins in AD pathogenesis. shown in Fig. 3C, which allows for peptide quantification in Cell Adhesion and Cytoskeleton and Membrane Traffick- both samples. It is worth noting that a trypsin auto-cleavage ing—A number of intercellular adhesion molecules have been peptide was used as an internal standard to normalize the shown to be localized in amyloid plaques in AD patients (40). experimental variation and to fit the elution time of selected We found that collagen I and fibrinogen were concentrated in peptides. The plaque/control ratio of A abundance was deter- the plaques. In addition to extracellular structures, integrity of mined to be around 80, indicative of the enormous enrichment the intracellular cytoskeleton is important for neuronal physi- of A in the plaques. The relative abundance of all other pro- ological functions such as axoplasmic flow of essential synaptic teins was quantified in a similar manner, resulting in the final components (41). In our study, all major isoforms of cytoskel- acceptance of 26 proteins enriched at least 2-fold in the plaques etal components, actin, tubulin, and neurofilament, were iden- of both AD cases (Table I). However, the majority of previously tified with high numbers of peptides, which indicates that they identified plaque proteins is not on this list because some are abundant species in the plaques as well as in non-plaque abundant proteins (e.g. actin and tubulin, Table S1) are present regions. Most interestingly, we identified one actin-binding but not necessarily concentrated in the plaque, and some low protein, coronin, concentrated in the plaque, although its po- abundance proteins (e.g. collagen XXV and antichymotrysin, tential role in pathogenesis remains unclear. Also enriched in Table S1) were detected only in one AD case perhaps due to plaques is the microtubule-associated protein tau that is more sensitivity limitation of the LC-MS/MS approach. Neverthe- commonly known to be associated with neurofibrillary tangles less, the list of 26 proteins that were identified and concen- and neurites (42) and likely represents the dystrophic neurite trated in the plaque regions in both AD cases are more likely to component in the plaques. The identification of a variety of be genuine plaque proteins. cytoskeletal protein elements, some of which are known to be Classification of Identified Plaque Proteins—To evaluate the relevant to AD, suggests that these elements may be involved proteins identified in the plaques from AD postmortem sam- in plaque formation, and cytoskeletal impairments may lead to Proteomic Characterization of Amyloid Plaques 37065 TABLE I List of 26 proteins enriched in amyloid plaques as compared with non-plaque areas The proteins and functional categories were sorted in the alphabetic order. When a peptide in the non-plaque control was not detected, we assumed the ratio was more than 2. Sometimes, even if a peptide ion was not sequenced by MS/MS, it was still possible to identify it in MS survey scan to enable quantification (see “Materials and Methods”). AD case 1 AD case 2 TM Protein name GenBank accession no. a b c Ctl Plaque P/C Ctl Plaque P/C Cell adhesion Collagen I, -1 polypeptide NP_000079.1 0 2 3.6 0 1 2 Fibrinogen,  NP_000500.1 0 3 20 2 2 Channels/receptors ATPase, Ca -transporting NP_001674.1 1 3 2.1 0 2 2 Chaperones Heat shock 90-kDa protein 1,  NP_031381.2 7 9 2.1 11 14 2.0 Cytoskeleton Coronin, actin-binding protein NP_009005.1 0 1 20 1 2 tau NP_058519.1 1 3 6.8 2 4 3.2 Inflammation Glial fibrillary acidic protein (GFAP) NP_002046.1 16 21 3.6 20 24 2.1 Vimentin NP_003371.1 4 12 9 11 15 2.3 Kinases/phosphatases and regulators 14-3-3 / NP_003395.1 2 4 3.1 5 8 3.5 14-3-3  NP_006752.1 2 5 6.1 7 9 4.4 14-3-3  NP_003397.1 4 6 7.7 5 10 7.4 Membrane trafficking Clathrin, heavy polypeptide 1 NP_001826.1 0 1 2 0 5 2.7 Dynamin 1 NP_004399.1 5 6 2.3 18 23 2.2 Dynein, heavy polypeptide 1 NP_001367.2 0 1 4.8 0 1 2 Metabolism Phosphofructokinase NP_002618.1 0 2 3.9 3 7 2.5 Others Amyloid -peptide NP_000475.1 1 1 80.0 0 1 2 Proteolysis Antitrypsin NP_000286.2 0 3 2 1 2 2.5 ATPase, H -transporting, lysosomal V0 subunit A NP_005168.2 0 1 2 1 3 2.4 ATPase, H -transporting, lysosomal V1 subunit B NP_001684.2 0 4 2.1 4 8 3.0 ATPase, H -transporting, lysosomal V1 subunit D NP_057078.1 0 1 2 1 3 2.6 ATPase, H -transporting, lysosomal V1 subunit E NP_001687.1 1 2 5.0 4 5 8.3 Cathepsin D NP_001900.1 0 1 20 3 2 Cystatin B NP_000091.1 1 2 2.9 0 1 2 Cystatin C NP_000090.1 0 1 20 1 2 Ubiquitin-activating enzyme E1 NP_695012.1 0 1 7.1 3 4 2.0 Vacuolar ATPase subunit H NP_057025.1 0 1 2 1 3 2.1 Ctl indicates the number of different peptides that identify a protein in the control samples of non-plaque regions. Plaque indicates the number of peptides that identify a protein in the plaque samples. P/C indicates the abundance ratio of proteins from the plaque samples versus non-plaque regions. a deficit in axoplasmic flow and eventually to neuritic dystro- ent in neurofibrillary tangles (46), and at least one 14-3-3 phy. Indeed, numerous proteins involved in membrane traffick- protein has further been shown to be an effector of tau phos- ing and protein sorting were revealed to be concentrated in the phorylation (47). Moreover, we attempted to detect protein plaques by the mass spectrometry analysis, such as clathrin phosphorylation sites in the plaque samples by tandem mass heavy chain, dynamin, and dynein heavy chain (Table I). This spectrometry, and we located two phosphorylated amino acid observation implicates that the AD plaque might sequester residues in neurofilament 3 (SPVPKSPVEEK and KAESPV- some key proteins to perturb the protein sorting system that is KEEAVAEVVTITK with modified sites shown in boldface with crucial for maintaining normal synaptic plasticity. underline). The first phosphorylation site was documented in Chaperones and Inflammation—It is well known that the tandem repeats in the neurofilament sequence and was exces- senile plaque core is surrounded by activated astrocytes, mi- sively modified in the AD brain (48). The second peptide indi- croglia, and dystrophic neurites (43, 44), and the heat shock cated a phosphorylation site on serine 736 that we show for the proteins exhibit high expression levels in reactive astrocytes in first time. These phosphorylation events may play a role in the areas rich in senile plaques (45). Consistently, we identified formation of dystrophic neurites surrounding the plaque core. many heat shock proteins (Table S1) and found that HSP90 Proteolysis—The ubiquitin-proteasome system plays a cru- was enriched in the plaques (Table I). We also identified glial cial role in the degradation of misfolded proteins and turnover fibrillary acidic protein and vimentin with high numbers of of cell signaling molecules (49). This study identified the ubiq- peptides; both are the major components of intermediate fila- uitin-activating enzyme enriched in the plaques. More strik- ments in activated glial cells. ingly, numerous subunits of lysosomal ATPase and cathepsin D Kinase/Phosphatase and Regulators—The imbalance of were found to be concentrated in the plaques, suggesting the phosphorylation/dephosphorylation activity is believed to con- high proteolytic activity in the plaques versus non-plaque re- tribute to AD pathology, as evidenced by tau hyperphosphory- gions. Moreover, we found in the plaques antitrypsin, cystatin lation in the neurofibrillary tangles. We identified multiple B, and cystatin C. Cystatin C is a cysteine proteinase inhibitor kinases (Table S1), but none of them were specifically enriched and has been shown to be up-regulated in AD and coaggregated in the plaque regions. Instead, three 14-3-3 isoforms showed a with A (50, 51). Cystatin C is also proposed as a potential risk significant degree of enrichment in the isolated plaques. Pre- factor for late-onset AD (52). Overall, the relative abundance of vious studies have demonstrated that 14-3-3 proteins are pres- proteolytic enzymes and inhibitors in multiple cellular proteo- 37066 Proteomic Characterization of Amyloid Plaques mice manifested high density of amyloid plaques, the majority of which is clearly surrounded by dynein immunoreactive structures that may represent enlarged neurites (Fig. 4F). Re- cently, missense mutations in dynein heavy chain have been genetically linked to progressive motor neuron degeneration and the formation of Lewy body-like inclusions (55). It has also been observed previously (56) that dynein immunoreactivity in AD brain tissue is significantly increased compared with nor- mal brain. Given that dynein is responsible for retrograde transport of vesicles along microtubules in the axon (57), the enrichment of dynein encircling the plaque core might trigger trafficking malfunction to cause neuritic dystrophy. DISCUSSION Our studies revealed a total of 488 proteins in amyloid plaques, representing three histopathological components: the plaque core, activated glial cells, and dystrophic neurites. Iden- tified intracellular proteins are likely derived from neurites and/or glial cells surrounding the plaque core, whereas extra- cellular proteins may be components of the plaque core. It is also possible that some intracellular proteins can leak out after plasma membrane damage during neuronal degeneration. By quantitative mass spectrometry, we further identified 26 pro- teins enriched in the plaques when comparing with the non- plaque control sample from the same AD case. This comparison is particularly valuable because both samples were derived from the same postmortem tissue specimen, which essentially eliminates the effects of many confounding factors (e.g. genetic variation, postmortem interval, etc.) that are often encountered FIG.4. Localization of plaque proteins by immunofluores- in human tissue studies. cence confocal microscopy. Sections of human postmortem AD neo- Our results confirmed the presence of most proteins that cortex were stained with A antibody (either polyclonal anti-A , A were detected previously in the plaques mainly by immunohis- and B, or monoclonal antibody 4G8, C and D, green) and antibodies tochemistry (40). The classic plaque components identified in against vimentin (A, red), Hsp70/90 complex (B, red), or dynein heavy this study include A, 1-antichymotrypsin (58), apolipopro- chain (C and D, red). Four serial confocal slices through a plaque region are shown in D ata1-m optical slice thickness and 4-m intervals tein E (59), collagen type XXV (60), cystatin C (61), -synuclein between slices. Brain sections containing cortex and hippocampus from (62), proteoglycans (63), and clusterin (64). On the other hand, nontransgenic (E) and PS1/APP double transgenic mice (F) at 9 months only a few proteins known to be amyloid plaque components of age were labeled with monoclonal A antibody 4G8 (green) and were missed in this study. Those included complement inhibi- polyclonal heavy chain antibodies (red). Scale bar, A, B, D, and the insert in F,50 m; C, E, and F, 100 m. tors (40), myeloperoxidase (5),  -macroglobin (65), superoxide dismutase (66), HO-1 (67), catalase (68), and cholinesterase lytic pathways strongly suggests the activation of protein deg- (69). The apparent absence of these proteins in the plaques in radation mechanisms and the interplay between proteolysis our study could be due to the low abundance of the proteins and inhibition activities during the plaque formation. and/or incompatibility of tryptic peptides with the LC-MS/MS Validation of Selected Plaque Components—To verify further system (12). Our results from two independent studies of AD the localization of some of the proteins identified, we undertook cases revealed that about two-thirds of the proteins were iden- immunostaining analysis on postmortem brain tissues for their tified in both cases. The difference between the two data sets colocalization with A. In Fig. 4A, monoclonal antibody against may be contributed by the sample variation in the patients and vimentin specifically labeled activated astrocytes surrounding the nature of shotgun proteomics strategy, as only a fraction of the fibrillary plaque core that was recognized by A antibody, peptides was selected and sequenced by mass spectrometry and some astrocyte processes were extended deeply into the when a complex peptide mixture was analyzed by the LC- plaque core, indicating a local inflammatory response in the MS/MS approach (29). plaque region (53, 54). Polyclonal antibodies against the Hsp70 Immunohistochemistry was employed as an independent ap- and Hsp90 complex show strong immunoreactive signals in proach to confirm the proteomic findings. Eight proteins (in dot-like structure located in the plaque regions (Fig. 4B), con- Table I and Table S1) were selected largely based on the avail- sistent with our proteomic analysis. More strikingly, dynein ability of the antibodies. Most of them are concentrated by heavy chain was detected as punctate and thread-like struc- varying degrees in the plaque regions, as exemplified by vimen- tures in many plaques labeled by A antibody (Fig. 4C), sug- tin, Hsp 70/90 complex, dynein (Fig. 4), -synuclein, glial fibril- gesting that dynein heavy chain is a constituent of plaque lary acidic protein, and synaptophysin (data not shown). Other neurites. Confocal microscopic images clearly indicated that proteins like actin and tubulin display a broad distribution but dynein heavy chain was enriched in the plaque, compared with are not specifically concentrated in the plaques (data not the surrounding neuropil (Fig. 4D). shown), although actin and tubulin are believed to be plaque The colocalization of dynein with amyloid plaque was corrob- components. Therefore, the list of 26 proteins according to orated in an AD model, PS1/APP double transgenic mice (36, quantitative analysis should contain much less false plaque 37). As expected, no plaque was visualized in 9-month-old constituents. control animals, and dynein heavy chain antibodies labeled the A is widely accepted as the major component of the amyloid soma and neurites in the cortex and hippocampus (Fig. 4E). In plaque core; consistently, our mass spectrometry analysis iden- great contrast, the similar brain regions in 9-month-old AD tified A in both plaque and non-plaque control but showed it Proteomic Characterization of Amyloid Plaques 37067 to be enriched about 80-fold in the plaques. No other proteins the extracted ion current of eluted peptides. More accurate were found to accrue at a similar level, suggesting that A is quantification of proteins could be achieved by applying stable the sole major protein species in the plaque core. Although A isotope labeling based techniques such as isotope-coded affinity aggregates are resistant to extraction by many reagents except tag strategy (73). To our knowledge, this is the first large scale analysis of for 70% formic acid (7), in order to extract other coaggregated proteins of interest, we chose 2% SDS among several tested proteins from AD amyloid plaques. The results of this study demonstrate that the protein molecules in amyloid plaques are extraction conditions because this buffer led to the best yield of highly complex and diverse, implicating the involvement of total proteins. As is well known, SDS dissolves A fibrils poorly many cellular pathways in disease development. The plaque (7), and accordingly, we identified only weak A immunoreac- subproteome identified in our study will be instructive for tivity in the SDS-solubilized plaque sample but not in the subsequent hypothesis-driven experiments on disease biomar- non-plaque control by Western blotting (data not shown). How- ker identification and molecular genesis of Alzheimer’s disease. ever, the small amount of A warranted strong signals in the LC-MS/MS analysis, which strongly suggested that many other Acknowledgments—We thank Dr. Michael Iuvone and James Wessel major protein aggregates in the plaques could barely elude for their help in LCM. We also thank Dr. Steven Gygi, Carson Thoreen, detection. and Rob Duarte for their help in computational analysis; Stephanie Carter for maintaining the animals; Sara Dodson for making mouse One major concern with this type of analysis is the purity of brain sections. In addition, we are grateful to Drs. Victor Faundez, the plaque sample, because LC-MS/MS can detect protein spe- John Wood, and Deanna Smith for providing antibodies. cies with high sensitivity. We used the LCM approach for the isolation of amyloid plaques from postmortem tissues stained REFERENCES by thioflavin-S. This dye preferentially labels proteins with 1. Hardy, J., and Selkoe, D. J. 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