Using the trypan blue staining, we examined the plasma membrane integrity of the cells following exposure to PSI for 48 h. Compared to the cells in dimethyl sulfoxide (DMSO) vehicle which were only shown as normal living cells excluding trypan blue stain (Figure 1A:a), the cells in PSI were shown as both abnormal living cells and dead cells, capable and incapable of excluding the dye, respectively (Figure 1A:b). Moreover, we quantitatively evaluated the decline of living cell density in the cells in PSI. The percentage of living cells differed significantly between the cells in the vehicle (100%) and PSI (85%), P < 0.05 (Figure 1, A: c).

Using the hematoxylin-eosin (H&E) histological staining, we detected the eosinophilic PSI-induced inclusions in the cells with exposure to PSI for 48 h. Compared to the cells in the vehicle which were shown without no morphological change in eosinophilic structure (Figure 1B:a), the cells in PSI were shown with distinct eosinophilic inclusions (Figure 1B:b). Further, similar to inducible inclusions from PC12 cells in PSI for 24 h or MES cells in mitochondrial respiratory chain complex I inhibitor, rotenone for 72 h [19,24], the PSI-induced inclusions appeared with a focal, homogeneous shape and displayed two types of morphology: nucleus-binding and nucleus-free inclusions. It was assumed that some of the PSI-induced inclusions were observed in the cytoplasm of the remaining cells, whereas the others were extruded into the extracellular space after destruction of the host cells [25].

At the first stage of isolation, using the H&E histological staining, we detected the eosinophilic PSI-induced inclusions, collected in the fraction of initial pellets. By homogenization of the cells with 0.5% NP-40 at 37°C for 30 min and subsequent centrifugation at 80 × g for 15 min, an abundance of intact PSI-induced inclusions (Figure 1B:c) was yielded. In practice, we also increased the number of nucleus-free PSI-induced inclusions. There was a significant difference in the percentage of the nucleus-free to total PSI-induced inclusions between the fraction of cells in PSI (51%; Figure 1B:d, Column 1) and the fraction of initial pellets (69%; Figure 1B:d,Column 2), *P < 0.05.

At the second stage of isolation, using the H&E histological staining, we first detected the PSI-induced inclusions, collected in the temporary and final fractions of resulting pellets. By four time-dependent incubations of the initial pellets with 200 U/ml DNase I at 37°C for different lengths of time during the 24 h period, the increasing number of nucleus-free PSI-induced inclusions were easily yielded at the cost of the gradual decline of nucleus-binding PSI-induced inclusions, and of which, when the one of the time-dependent incubations ran out for the entire 24 h, the increase of nucleus-free PSI-induced inclusions instead of the decline of nucleus-binding PSI-induced inclusions could be most easily yielded. In detail, after the four time-dependent incubations for 1 h (Figure 1B:e), 6 h (Figure 1B:f), 12 h (Figure 1B:g) and 24 h (Figure 1B:h), the inclusive regions of nucleus (close arrow heads) but not the inclusive regions of inclusion (open arrows) within the nucleus-binding PSI-inclusions were progressively reduced. The percentage of the nucleus-free to total PSI-induced inclusions in the temporary fraction of resulting pellets which was collected after the one of the four time-dependent incubations of the initial pellets for 1 h (79%; Figure 1B:j Column 3) differed significantly from each of the percentages in the temporary fractions of resulting pellets which were collected after the other three of the four time-dependent incubations of the initial pellets for 6 h (80%; Figure 1B:j Column 4), 12 h (81%; Figure 1B:j Column 5) and 24 h (93%; Figure 1B:j Column 6), *P < 0.05. Interestingly, by the incubation of the initial pellets with 200 U/ml DNase I and 250 μg/ml RNase A for 24 h, a large quantity of nucleus-free PSI-induced inclusions instead of few nucleus-binding PSI-induced inclusions was observed in the final fraction of resulting pellets (Figure 1B:i). All percentages in the first four temporary fractions of resulting pellets (Figure 1B:j, Column 3-6) differed significantly from the percentage in the final one (97%; Figure 1B:j Column 7), **P < 0.05.

At the second stage of isolation, we detected the alpha synuclein (alpha-SYN) positive, eosinophilic inclusions, collected in the isolated fraction of PSI-induced inclusions, using the alpha-SYN immunostaining and the H&E histological staining, and then detected the relatively small amount of histone H1 present in the same fraction using the West blotting for histone H1, one of the most abundant proteins in the nucleus [26,27]. In general, subcellular particles, such as cellular debris and technical artifacts, were created after the homogenization at the first stage of isolation, and in particular, nuclear proteins were remained after the nucleus degradation at the second stage of isolation. Thus, by centrifuging the final fraction of resulting pellets in discontinuous Percoll-mediated 12%-35% gradients at 35,000 × g for 30 min, the intact PSI-induced inclusions could be easily divided from these subcellular particles and collected as preparation of the target fraction. On one hand, whether the PSI-induced inclusions were formed in the cells in PSI (Figure 1C:a and 1C:a') or isolated through cellular fractionation (Figure 1C:b and 1C:b'), they were stained as alpha-SYN positive, eosinophilic structures. On the other hand, in comparison to the intensive signal of blotted histone H1 in gel image and its high level in the column diagram, both of which were characteristic of the large amount of nuclear proteins for the samples of the cells in the vehicle or in PSI, the faint signal of blotted histone H1 in gel image and its low level in column diagram were characteristic of the trace of nuclear proteins for the sample of the isolated fraction of PSI-induced inclusions (Figure 1D:a and 1D:b). In the end, the pure intact PSI-induced inclusions were found in the isolated fraction.

We detected the protein spot pattern of the isolated fraction of PSI-induced inclusions using 2-D gel electrophoresis. Since aggresomes in cell culture models of PD act as disposal grounds for insoluble protein aggregates [4,11], we assessed proteome organization of the isolated fraction of PSI-induced inclusions by systematic analysis of protein complexes. Lysis buffer was added to the isolated fraction of PSI-induced inclusions to split inclusions, and an optimal formulation of rehydration solution was used to dissolve proteins from the split-products [28,29]. In conventional ultrasonic extraction, the enhancement of extraction efficiency is attributed to acoustic cavitation [20,30]. Thus, soluble proteins dissolved from the isolated fraction of PSI-induced inclusions were successfully resolved in 2-D gel as many individual protein spots. As with the two samples of proteins extracted from the homogenates of the cells in the vehicle and in PSI (Figure 2A:a and 2A:b), the sample of proteins extracted from the isolated fraction of PSI-induced inclusions was resolved on the left and central regions on 2-D gel (Figure 2A:c). That is the proteins displayed here are acidic and neutral proteins, as consistent with a previous observation that nervous system proteins in rat are mainly composed of acidic and neutral proteins [31]. In addition, most of the resolved proteins were within the molecular weight range of protein markers or the pH scale of isoelectric points (pI) or both of them. Undoubtedly, the proteomic profiling of the soluble proteins extracted from the isolated fraction was represented by the specific protein spot pattern in 2-D gel. Of the protein spots that were better focused in at least three of the four gels, one hundred and fourteen spots were reproducibly observed. Each spot was found not to differ significantly in terms of appearance, disappearance and shift. These protein spots of interest were selected for identification via peptide mass fingerprinting (PMF) (Figure 2A:d).

It is pointed out that values of both molecular mass and pI supplied in a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) data file are usually theoretical. Some of the spots identified as proteins were found to alter their positions across 2-D gel in the present experimental condition. In general, the observed difference most probably reflects mechanisms for the augmentation of ubiquitin fusion-protein expression in eukaryotes when proteasomes are inhibited beyond a threshold level [32-34].

We detected experimental PMF spectra of the target protein spots using MALDI-TOF MS, and then PMF data for protein identification using two on-line proteomic search engines. Preliminarily, a total of fifty six proteins were identified after submission of their PMFs to the ProPound search engine against the single NCBInr database (shown in Additional file 1). Furthermore, forty eight of the fifty six proteins were consistently identified after submission of their PMFs to the Mowse score-based Mascot search engine against the multiple NCBInr, SwissProt, MSDB databases (shown in Additional file 1). For example, prolyl-4-hydroxylase beta subunit (P4HB) identified via its PFM against the single database is shown with a group of MS data, such as 32.4% sequence coverage, 0.000 expectation, 8 matched peptides and theoretic sequences of the matched peptides (Figure 2B:a and 2B:b). After consistent identification via its PFM against the multiple databases, it was shown with its three comparable score values, viz., a top score value of 71 (> 61) compared to a runner up value of 38 (< 61) for NCBInr branch, 71 (> 51) compared to 37 (< 51) for SwissProt branch, and 71 (> 56) compared to 38 (< 56) for MSDB branch (Figure 2B:c to 2B:e). The protein was ultimately determined.

Among the fifty six proteins, twenty were found to be previously reported components of LBs. These components of LBs identified in the isolated fraction of PSI-induced inclusions are listed as follows: two key proteins in the synthesis of neural transmitter dopamine (DA), i.e. tyrosine hydroxylase (TH) and 14-3-3epsilon (No.1-2) [35,36]; two microtubulins, i.e. tubulin alpha1 and tubulin beta5 (tubb5) (No.8-9) [37]; two mitochondria matrix proteins, i.e. ATP synthase beta subunit (F1-ATPase beta) and ubiquinol-cytochrome c reductase core protein 1 (Uqcrc1) (No.20-21) [38,39]; Cu, Zn superoxide dismutase (C, Z-SOD; No.22) [40,41]; six proteasomal subunits, i.e. proteasome subunit beta 5, proteasome 26 S subunit ATPase 2 (PSMC2), proteasome 26 S subunit ATPase 5, proteasome 26 S subunit ATPase 6, proteasome 26 S subunit non-ATPase 11 and proteasome 26 S subunit non-ATPase 13 (No.26-31) [23,42]; and seven chaperone proteins, i.e. 27-kD heat shock protein 1 (HSP27; No.32) [43], heat shock protein 32 (HSP32; No.33) [44], 70-kD heat shock protein 1A/1B (HSP70; No.35) [23], 71-kD heat shock cognate protein (HSC70; No.36) [11], 14-3-3zeta (No. 43) [45,46], T-complex polypeptide 1 beta subunit (TCP-1beta; No.46) [47] and Valosin-containing protein (No.48) [48]. Comparatively, the twenty eight proteins were newly identified and, if selected for further study, may provide novel clues to our understanding of the potential proteomic candidates of aggresomes in response to proteasome inhibition by PSI. For the other eight proteins which were incompletely determined by the Profound search engine using the single NCBInr database alone (shown in Additional file 2), maybe it's preferable for them to be identified in the future since update on construction of a protein reference database has been considered as a discovery resource for proteomics [19-21]. These proteins, most of which were recognized as a profile of proteins within cellular processes mediated by the UPS, a profile of constituents of the UPS and a profile of chaperone proteins, are classed into at least nine accepted categories [4,19,20]. Functional classification of the fifty six proteins and their percentages are shown in Figure 3. Given that in eukaryotes, aggresomes sequester aberrant and potentially cytotoxic proteins from the a wide range of cellular processes mediated through the UPS and also serve as sites for the recruitment and concentration of constituents of the UPS and chaperone proteins [11], to date the protein dataset are at the level of proteomics valuable for characterization of aggresomes, induced in PC12 cells by PSI and also in vitro by many other proteasome inhibitors such as lactacystin or MG132 [4,14]. Moreover, the profile of chaperone proteins, accounting for the predominant portion of the protein dataset and classed into the category of protein folding and transport, have been found in available literatures to share some similarity between protein families and specialize in various cellular localization resources (summarized in Additional file 3). They are at the level of bioinformatics valuable for providing cellular information on relationship between chaperone proteins, known as aggresome related proteins, and aggresomes, known as intermediate compartments [11].

Proteins identified via PMF need validation to show their biological presence. A candidate selected for validation was determined by three criteria as follows: it was significantly focused as an individual spot in 2-D gel and detected as with a desirable group of MS identification data; it has not been reported in published literature on LBs; it matches to commercial specific antibody. Thus, we sought to determine whether P4HB, a member of the protein disulfide isomerase (PDI) family in endoplasmic reticulum (ER) lumen, was present in PSI-induced inclusions in PC12 cells with proteasome inhibition, and if so, whether it redistributed from its cellular location resource during the induction of inclusions. We validated P4HB in the developmental process of PSI-induced inclusions in PC12 cells in PSI using the P4HB immunostaining and the H&E histological staining. Compared to the uniform immunostaining throughout cytoplasm of the cells in the vehicle (Figure 4A), P4HB tended to converge to juxtanuclear regions in the cells in PSI (Figure 4B to 4E). Specifically, after proteasome inhibition by PSI for 12 h, P4HB was present with immunostaining of asymmetric structures throughout cytoplasm and around nuclei (Figure 4B); after the proteasome inhibition for 24 h or 36 h, P4HB was present with immunostaining of granular and elliptical perinuclear structures (Figure 4C and 4D); after the proteasome inhibition for 48 h, P4HB was present with immunostaining of spherical structures (Figure 4E), which was also shown in the isolated fraction of PSI-induced inclusions (Figure 4F). Further, compared to hematoxylin staining of ovoid nuclei of the cells in the vehicle (Figure 4A'), P4HB-captured structures stained readily with eosin were present in the cells in PSI for the different lengths of time (Figure 4B' to 4E') and in the fraction of PSI-induced inclusions isolated from the cells in PSI for 48 h (Figure 4F').

Unless specified otherwise, analytical reagents and reagents of cell culture grade were purchased from Amersham Biosciences (Uppsala, Sweden). PSI, N-benzyloxycarbonyl-Ile-Glu (O-t-butyl)-Ala-Leu-al, was from EMD Biosciences (an affiliate of Merck Chemicals, Darmstadt, Germany). Cell culture plastics, culture media and supplying chemicals were from Gibco (Grand Island, NY, USA). DNase I and RNase A were from TaKaRa Biotechnology (Dalian, China). Percoll (density 1.131 g/ml) and proteinase inhibitors [4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin] were from Sigma (St. Louis, USA). Mouse anti-rat alpha-SYN monoclonal antibody (4D6: sc-65500) and mouse anti-rat histone H1 monoclonal antibody (sc-56694) were from Santa Cruz Biotechnology (California, USA); mouse anti-rat P4HB monoclonal antibody (MAB2073) was from Chemicon (Fuji, Japan). Cy3-conjugated goat anti-mouse secondary antibody (00009-1) was from Proteintech Group (Chicago, USA).

PC12 cells were purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China), and their culture was carried out as described elsewhere [25].

PC12 cells, seeded at a density of 1 × 10⁵ cells/ml in 96-well plates and incubated for 24 h, were exposed to 10 μM PSI or equal volume of its vehicle, 0.1% DMSO (V/V) in culture medium, for 48 h [25]. Then, they were centrifuged at 1000 rpm (or 93 × g), 4°C for 5 min [101] and stained with 50 μl of 1% trypan blue (W/V) for 2 min [102]. After supernatant was removed from the cell cultures, living cells at a density of at least 100 cells per field were viewed under an upright fluorescence microscope with IF550 green interference filter in the light path. The number of living cells was counted in nine random fields (three fields per well of a 96-well plate, three different wells) and expressed as a percentage [103,104]. Chi square test (χ² test) was used to determine whether a percent difference was significant between the cells without and with PSI exposure. Probability value (P) < 0.05 was accepted as significant. Measurements were repeated at least 3 times.

Formation of PSI-induced inclusions and their isolation through cellular fractionation were carried out as described elsewhere [25]. Cells exposed to 10 μM PSI were subjected to the H&E histological staining to monitor appearance of the PSI-induced inclusions. Progress in the isolation of PSI-induced inclusions included two sequential stages: separation for an abundant fraction of intact PSI-induced inclusions, and purification for preparation of the target fraction. At the first stage of isolation, briefly, cells in PSI for 48 h were scraped, collected and washed in ice-cold Tris-buffered saline solution (TBS; pH 7.4) and ice-cold 8% sucrose (W/V) in 0.1 × TBS one after the other. The fraction of the cells was frozen repeatedly with liquid nitrogen, and homogenized with a certain amount of lysing buffer consisting of 1 mM Hepes (pH 7.2), 0.5 mM MgCl₂, 0.5% NP-40 (V/V), 0.1% beta mercaptoethanol (V/V), and 1% proteinase inhibitors (V/V). After repeated suspension and vibration, the lysate was incubated at 37°C for 30 min until the cells were thoroughly homogenized. Initial pellets were collected by low speed centrifugation at 80 × g for 15 min, washed in ice-cold buffer solution L consisting of 1 mM Hepes, 0.5 mM MgCl₂ and proteinase inhibitors, and recollected by centrifugation at 836 × g.

At the second stage of isolation, the fraction of initial pellets was subjected to the H&E histological staining to monitor presence of the eosinophilic PSI-induced inclusion. In the next isolation, four equal aliquots removed from the fraction of initial pellets were incubated in 200 U/ml DNase I alone in DNase I buffer plus proteinase inhibitors at 37°C for 1 h, 6 h, 12 h and 24 h, respectively, whereas another equal aliquot from the fraction was incubated in the DNase I solution with addition of 250 μg/ml RNase A at 37°C for 24 h. In their respective incubations, initial pellets were repeatedly suspended and vibrated vigorously. After incubation, the first four temporary fractions of resulting pellets and the final one were collected by centrifugation at 836 × g, washed with 0.32 M sucrose in TBS, and subjected to the H&E histological staining to monitor presence of the eosinophilic PSI-induced inclusion.

The final fraction of the resulting pellets was diluted to 600 μl using 12% Percoll in TBS (V/V) and overlaid on equal volume of 35% Percoll in TBS. The material band just below the interface of the sample and 35% Percoll was removed, collected by centrifugation at 35,000 × g, washed in 250 mM sucrose in 10 mM Tris (pH 8.0), and recollected by centrifugation at 4000 × g. This isolated fraction of PSI-induced inclusions was the preparation of target fraction. The target fraction was subjected to the α-SYN immunostaining and the H&E histological staining to monitor presence of the PSI-induced inclusion, and then subjected to the Western blotting for histone H1 to monitor contamination of nuclear protein content.

Eosinophilic PSI-induced inclusions were examined by an experienced observer not familiar with the sample identity. The cells exposed either to the vehicle or to PSI, the fraction of initial pellets, and the five fractions of resulting pellets were all subjected to the H&E histological staining, as previously described [25]. The number of PSI-induced inclusions was counted in nine random fields (three fields per section, three different sections on slide), and expressed as a percentage of nucleus-free to total PSI-induced inclusions. The χ² test was used to determine whether there was significant difference in the percentage between every two objects of comparison as follows: between the cells in PSI and the fraction of initial pellets, between the first one temporary fraction of resulting pellets and each of the other three temporary fractions of resulting pellets, and between each of the four temporary fractions of resulting pellets and the final fraction of resulting pellets. P < 0.05 was accepted as significant. Measurements were repeated at least three times.

The isolated fraction of PSI-induced inclusions, together with a control (cells in PSI), was first subjected to the alpha-SYN immunostaining and then the H&E histological staining. In the application of immunostaining for alpha-SYN, briefly, the isolated fraction of PSI-induced inclusions was spun on slides and fixed in 4% paraformaldehyde (W/V). Sections on the slides were permeabilized with phosphate-buffered saline containing Triton X-100 (PBS-T; V/V), blocked in 5% bovine serum albumin (BSA; V/V) in PBS for 1 h, incubated with primary antibody at 1:50 dilution in PBS at 37°C for 3 h and incubated with secondary antibody at dilution 1:100 in PBS-T at 37°C for 3 h, and mounted with 80% glycerol (V/V). The dilutions of primary and secondary antibodies were according to the reference to manufacturer's instructions and the description in literature [105]. Control sections were examined, as described above, excluding primary antibody in PBS. Measurements were repeated at least three times.

The isolated fraction of PSI-induced inclusion, together with two controls (cells in PSI and in the vehicle), was subjected to the Western blotting for histone H1. Briefly, the isolated fraction of PSI-induced inclusion was frozen repeatedly with liquid nitrogen, and dissolved with a certain amount of lysing solution consisting of 30 mM Tris, 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) -dimethylammonio]-1-propanesulphonate (CHAPS; W/V), 60 mM dithiothreitol (DTT), and proteinase inhibitors. The mixture was incubated, concussed, and collected by centrifugation at 25,000 × g, 4°C for 30 min. Approximately 20 μg of protein extracts adjusted to a total volume of 20 μl was resolved by polyacrylamide gel electrophoresis in a gel buffer. A 0.5-mm-thick discontinuous polyacrylamide gel system without stacking gel layer was determined by 0.1% sodium dodecyl sulfate (SDS; W/V), 15% total gel (T; W/V), and 2.6% cross-linking agent (C; W/W). Protein markers with a range of 23-132 kD were used to measure molecular masses of the resolved proteins. After the gel was transferred to a polyvinylidene difluoride membrane, the membrane was blocked in 5% BSA in PBS-T at 37°C for 1 h, incubated with primary antibody at 1:100 dilution in 1% BSA in PBS-T at 4°C overnight and incubated with secondary antibody at 1:500 dilution in 1% BSA in PBS-T at 37°C for 2 h. The blotted membrane was scanned using a Typhoon 9400 fluorescent laser scanner (Emission filter at 580 BP Cy3 mode, wavelength at 532 nm; Amersham Biosciences) with Typhoon Scanner Control Version 3.0 (Amersham Biosciences). The digitalized image of the immunoblot was analyzed with DeCyder Image QuantTM Version 5.0 (Amersham Biosciences). Bands of histone H1 in image were densitometrically quantified using ImageQuant Version 5.0 (Amersham Biosciences). Dilutions of both primary and secondary antibodies were according to the reference to manufacturer's instructions and the description in literature [26,27]. Control blotting membrane was carried out, as described above, excluding primary antibody in 1% BSA in PBS-T. Measurements were repeated at least three times.

Protein extraction, 2-D gel electrophoresis and image analysis were carried out as described elsewhere [25,106]. Briefly, the isolated fraction of PSI-induced inclusion, frozen repeatedly with liquid nitrogen, was dissolved with a lysis buffer consisting of 30 mM Tris, 7 M urea, 2 M thiourea, 4% CHAPS, 60 mM DTT, 2% pharmalyte (Ph 3.0-10.0; V/V) and proteinase inhibitors. The mixture was incubated, sonicated, and collected by centrifugation at 25,000 × g, 4°C for 30 min. A certain amount of protein extracts prepared from the isolated fraction of PSI-induced inclusion, together with two controls prepared from cells in PSI and the vehicle, were demineralized with 2-D Clean-Up kit, re-dissolved with a rehydration solution consisting of 8 M urea, 2% CHAPS (W/V), 0.6% DTT (W/V), and 0.5% immobilized pH gradients buffer (V/V), and applied to a non-linear Immobiline™ DryStrip IPG gel strip (pH 3.0-10.0; 24 cm; Amersham Biosciences). The first dimension electrophoresis was run on an Ettan IPGphor II Isoelectric Focusing Unit (50 μA per strip; Amersham Biosciences). The strip was then equilibrated in a loading solution consisting of 50 mM Tris-HCl (pH 8.8), 6 M urea, 2% SDS, 30% glycerol, 2% DTT and a trace of bromophenol blue, re-equilibrated in the same loading solution including 4% (W/V) iodoacetamide but excluding DTT, and placed onto the top of polyacrylamide gel (24 × 20 cm²). A 1-mm-thick continuous polyacrylamide gel system without stacking gel layer was determined by 0.1% SDS, 12.5% T, and 2.6% C. The protein markers were used to mark positions of proteins resolved in gel. The second dimension electrophoresis was run on an Ettan DALT six electrophoresis unit (2 W per gel, 600 V, 400 mA; Amersham Biosciences). Coomassie blue-stained gels were scanned using an Umax CE scanner (Amersham Biosciences) with Image Master Labscan version 3.01b (Amersham Biosciences). The digitalized image of the gels was analyzed with Image Master 2-D Evolution version 2003.02 (Amersham Biosciences). Spots in images were densitometrically quantified and statistically evaluated by pattern analysis of computer-assisted pit assessment to detect whether spots appeared reproducibly significant in at least 3 of the 4 gels. These protein spots of interest were selected for MS analysis. If a spot was not detected in at least 3 of the 4 gels, it was considered to be negligible in the present experimental condition.

In-gel digestion, MS and database search were carried out as described elsewhere [25,107]. Briefly, using an Ettan Spot Picker robotic workstation (Amersham Biosciences), the protein spots of interest were automatically selected, excised and placed in 96-well plates at a gel plug per well. Using an Ettan TA Digester robotic workstation (Amersham Biosciences), the gel plugs were twice destained, dehydrated, desiccated and incubated in 10 μl of 1 μg/μl modified porcine trypsin at 4°C overnight, and after which the digested peptide fragments were extracted twice and the two combined extracts were desiccated in other 96-well plates. Using an Ettan Spotter robotic workstation (Amersham Biosciences), 0.3 μl of the digested peptide fragments and an equal volume of 4 μg/μl α-cyano-4-hydroxy-transcinnamic acid were deposited on the surface of a sample slide (Amersham Biosciences). The pre-mix was allowed at room temperature to facilitate uniform dispersion of the digested peptide fragments in a large mix.

Signals of MS were recorded in the positive ion reflection mode on a MALDI-TOF Pro workstation (Amersham Biosciences). The preset threshold values suitable for acquiring and processing MS were referred to our previous requirements [25]. Additionally, the most intensive signals of MS had to be characterized by at least 3 to 5 strongest peaks. Such an additional criterion can efficiently distinguish the target protein from the interference background, since experimental spectra for protein identification consist of the mass and intensity pairs, one pair for each peak [108].

Protein identification via PMF was initially accomplished using the ProFound search engine against the latest version of the single NCBInr database (fully integrated and included in the Pro workstation or at http://prowl.rockefeller.edu/prowl-cgi/profound.exe), and was further enhanced using Mowse score-based Mascot search engine against the latest version of the multiple NCBInr, SwissProt, MSDB databases http://www.matrixscience.com/search_form_select.html. In the initial identification, the requirements of evaluation standard were allowed as follows: sequence coverage of at least 20%, expectation as much as 0.05, a set of at least 5 matched peptides, and a gap of at least 3 peptides between the accepted protein candidate and the first excluded one in the list of protein candidates provided by the single NCBInr database [25]. For the enhancement of identification, except that mass tolerance at ± 0.2 D as a preset start-up value of searching parameter for the single database is different from mass tolerance at ± 0.1 D for the multiple databases, the others were identical in the two types of protein databases [25]. A protein, if assigned as the identical protein candidate against the two types of protein databases, was ultimately determined.

Cells exposed to PSI for 12, 24, 36 and 48 h, respectively, and a fraction of inclusions isolated from cells in PSI for 48 h were first subjected to the P4HB immunostaining and then the H&E histological staining. The dilutions of both primary (1:40) and secondary (1:100) antibodies were carried out according to the reference to manufacturer's instructions and the description in literature [109].