Multiple Protein Analysis of Formalin-fixed and Paraffin-embedded Tissue Samples with Reverse phase Protein Arrays

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.Many diseases are characterized by the expression of specific proteins and the activation status of distinct signaling pathways (1). Thus, protein expression profiling and activation patterns are instrumental for understanding disease, the development of effective treatments, and the identification of patients who will respond to particular therapies. Traditional ways of analyzing protein expression (e.g. Western blot) can be used for these purposes but often are labor intensive, have low throughput, and consume high sample volumes. Reverse-phase protein array (RPPA)¹ technology is a very promising method that circumvents these issues (2–4). For RPPA, minute amounts of whole protein lysates from a multitude of samples are spotted onto slides, and individual proteins are detected via protein-specific antibodies. This enables medium- to high-throughput analysis of precious low-volume sample material.Lysates for RPPA have so far been generated mainly from cell lines or fresh frozen tissue. However, because of the high amount of effort involved in the use of liquid nitrogen for sample preservation, in almost all hospitals worldwide formalin fixation and paraffin embedding is the preferred method for tissue preservation. Therefore, it would be highly desirable if protein-specific epitopes could be quantitatively extracted and analyzed from formalin-fixed and paraffin embedded (FFPE) tissue, as this would make the majority of clinical specimens accessible for mechanistic protein-based research.In recent years, several research groups have established protocols for protein extraction from FFPE tissue. Common to all of them is the use of high concentrations of ionic detergents, such as sodium dodecyl sulfate, and high temperature. It was shown that these methods even make it possible to extract full-length proteins from FFPE tissue (5–12). The coefficient of variation of the relative extraction efficiency based on Western blot and densitometric assessment of actin typically is below 20% (13). To assess whether the analysis of FFPE tissue lysates would result in biologically meaningful data, we analyzed FFPE breast cancer tissue samples by RPPA for the expression of human epidermal growth factor receptor 2 (HER2) and compared it to HER2 assessment by the gold standard used in clinical practice, which is based on immunohistochemistry (IHC). Successful recovery of HER2 from FFPE tissue should result in concordant HER2 classification between RPPA and IHC.In the second part of the study, FFPE samples of non-small cell lung cancer (NSCLC) were examined via RPPA. Samples from two subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SCC), were analyzed for more than 150 proteins, including two proteins that are known to be differentially expressed between the two subtypes. The objectives of this analysis were to further assess the validity of the approach by confirming the two positive controls and to identify new markers for the differentiation of the two subtypes of NSCLC.     

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer''s disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson''s disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states.The mechanistic/mammalian target of rapamycin complex 1 (mTORC1)¹ is a serine/threonine protein kinase that is highly expressed in many cell types (1). In the brain, mTORC1 tightly coordinates different synaptic plasticities — long-term potentiation (LTP) and long-term depression (LTD) — the molecular correlates of learning and memory (2–5). Because mTORC1 is at the core of many synaptic signaling pathways downstream of glutamate and neurotrophin receptors, many hypothesize that dysregulated mTORC1 signaling underlies cognitive deficits observed in several neurodegenerative diseases (3, 6–17). For example, mTORC1 and its downstream targets are hyperactive in human brains diagnosed with Alzheimer''s disease (AD) (18–20). Additionally in animal models of autism spectrum disorder (ASD), altered mTORC1 signaling contributes to the observed synaptic dysfunction and aberrant network connectivity (13, 15, 21–27). Furthermore, epilepsy, which is common in AD and ASD, has enhanced mTORC1 activity (28–32).Phosphorylation of mTORC1, considered the active form, is generally regarded to promote protein synthesis (33). Thus, many theorize that diseases with overactive mTORC1 arise from excessive protein synthesis (14). Emerging data, however, show that suppressing mTORC1 activation can trigger local translation in neurons (34, 35). Pharmacological antagonism of N-methyl-d-aspartate (NMDA) receptors, a subtype of glutamate receptors that lies upstream of mTOR activation, promotes the synthesis of the voltage-gated potassium channel, Kv1.1, in dendrites (34, 35). Consistent with these results, in models of temporal lobe epilepsy there is a reduction in the expression of voltage-gated ion channels including Kv1.1 (30, 31, 36). Interestingly in a model of focal neocortical epilepsy, overexpression of Kv1.1 blocked seizure activity (37). Because both active and inactive mTORC1 permit protein synthesis, we sought to determine the proteins whose expression is altered when mTORC1 phosphorylation is reduced in vivo.Rapamycin is an FDA-approved, immunosuppressive drug that inhibits mTORC1 activity (38). We capitalized on the ability of rapamycin to reduce mTORC1 activity in vivo and the unbiased approach of mass spectrometry to identify changes in protein expression. Herein, we provide evidence that mTORC1 activation bidirectionally regulates protein expression, especially in the PSD where roughly an equal distribution of proteins dynamically appear and disappear. Remarkably, using protein–protein interaction networks facilitated the novel discovery that PARK7, a protein thus far only implicated in Parkinson''s disease, (1) is up-regulated by increased mTORC1 activity, (2) resides in the PSD only when mTORC1 is active, and (3) is aberrantly expressed in a rodent model of TSC, an mTORC1-related disease that has symptoms of epilepsy and autism. Collectively, these data provide the first comprehensive list of proteins whose abundance or subcellular distributions are altered with acute changes in mTORC1 activity in vivo.     

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R²: 0.99–1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.MS based proteomics has traditionally been used to investigate complex biological systems, such as cell lines, plasma, or fresh-frozen tissues (1, 2). In the last decade however, MS proteomics has extended to the analysis of formalin-fixed paraffin-embedded (FFPE)¹ tissues (3). Formalin fixation is the gold standard for sample storage in clinical pathology because it allows optimal preservation of the morphological features of the tissue and it is economically attractive (storage at room temperature over several years or decades) (4). Several studies have shown that although the individual peptides retrieved and identified from fresh-frozen and FFPE tissues may differ, the biological information obtained from both types of material in terms of number of proteins identified, cellular location and molecular function is very similar (5–10). A number of proteomics studies were reported, which used untargeted MS on FFPE tissues to compare diseased and healthy samples in the search for potential novel biomarkers (10). Nevertheless, these untargeted MS workflows do not allow performing accurate protein quantification on large numbers of samples. One option is to use targeted MS approaches, such as selected reaction monitoring (SRM), which are highly quantitative and reproducible over many samples (11, 12). Additionally, SRM assays allow a high level of multiplexing (several hundreds of peptides can be measured in parallel in a single analysis) (13). The lack of access to a sufficient number of high-quality samples annotated with comprehensive clinical data sets may be a limiting factor for preclinical exploratory phase biomarker studies (14). The possibility to use FFPE samples for MS-based proteomics, in particular for quantitative targeted approaches, would therefore open tremendous perspectives for performing large retrospective biomarker discovery and verification studies. Indeed, in addition to being widely available, most FFPE tissues are annotated with clinical data. Moreover, targeted MS workflows applied to FFPE samples are complementary to techniques requiring high-quality antibodies, such as immunohistochemistry (IHC) or reverse-phase protein arrays (RPPA). These techniques all rely on the measurement of the target protein, with SRM measuring one or ideally several peptides as surrogates of the protein (15, 16). In opposition to IHC and RPPA however, SRM does not rely on the presence of a specific antibody for analyte detection, thereby avoiding cross-reaction issues and making assay development relatively rapid and cost effective. Although SRM is less advanced for protein analysis than for small molecules quantification, the technique was demonstrated to be selective, reproducible, and highly quantitative over large dynamic ranges for proteins as well (17–19). However, although the equivalence of qualitative analyses performed on fresh-frozen and FFPE samples has been investigated and demonstrated, only a few studies evaluated quantitative targeted MS approaches in FFPE samples (20, 21). Targeted proteomics performed on FFPE tissues is still in its early days and known limitations of this technique include the loss of morphologic features of the tissue and an extensive sample preparation, causing a low sample throughput (20). Moreover, targeted proteomics quantifies peptides as surrogates of a protein, with the former not necessarily agreeing in absolute terms with the latter. This is true for bottom-up proteomics in general, but it is of particular importance for FFPE tissues.In this study, we critically assessed the validity of targeted MS applied to peptide quantification in FFPE tissues. We developed and evaluated an SRM assay for the quantification of six peptides of the human receptor tyrosine-protein kinase erbB-2 (HER2) and compared the obtained results with those of standard methods used in clinical pathology, namely IHC and fluorescence in situ hybridization (FISH). HER2 was chosen as a candidate protein because its overexpression is routinely assessed in breast tumors in order to determine susceptibility to anti-HER2 treatment (22). Depending on the laboratory, HER2 overexpression can be assessed by IHC or the amplification of the corresponding gene (ERBB2) can be quantified by FISH (23). Several laboratories use both techniques for decision-making, as they are complementary.In a first step, we developed an SRM assay for the quantification of HER2 peptides in archival clinical FFPE tumor tissues and assessed its analytical performance, including linearity, precision and lower limit of quantification (LLOQ). We then demonstrated the applicability of the method by quantifying HER2 peptides in a cohort of 40 FFPE tumor tissues expressing different levels of HER2 (selected based on ERBB2 gene amplification status). The samples originated from surgical resections performed on women with invasive mammary carcinomas. We thereby investigated several options to normalize the results in regard to sample size. Finally, in order to confirm the validity of SRM as a suitable method for protein quantification in FFPE tissues, we determined the agreement between data generated by SRM and data generated by IHC or FISH.     

Matricellular Protein CCN3 (NOV) Regulates Actin Cytoskeleton Reorganization

CCN3 (NOV), a putative ligand for integrin receptors, is tightly associated with the extracellular matrix and mediates diverse cellular functions, including cell adhesion and proliferation. CCN3 has been shown to negatively regulate growth although it promotes migration in a cell type-specific manner. In this study, overexpression of CCN3 reduces growth and increases intercellular adhesion of breast cancer cells. Interestingly, CCN3 overexpression also led to the formation of multiple pseudopodia that are enriched in actin, CCN3, and vinculin. Breast cancer cells preincubated with exogenous CCN3 protein also induced the same phenotype, indicating that secreted CCN3 is sufficient to induce changes in cell morphology. Surprisingly, extracellular CCN3 is internalized to the early endosomes but not to the membrane protrusions, suggesting pseudopodia-enriched CCN3 may derive from a different source. The presence of an intracellular variant of CCN3 will be consistent with our finding that the cytoplasmic tail of the gap junction protein connexin43 (Cx43) associates with CCN3. Cx43 is a channel protein permitting intercellular communication to occur. However, neither the channel properties nor the protein levels of Cx43 are affected by the CCN3 protein. In contrast, CCN3 proteins are down-regulated in the absence of Cx43. Finally, we showed that overexpression of CCN3 increases the activity of the small GTPase Rac1, thereby revealing a pathway that links Cx43 directly to actin reorganization.The CCN (CYR61/Connective Tissue Growth Factor/Nephroblastoma Overexpressed) family of multimodular proteins mediates diverse cellular functions, including cell adhesion, migration, and proliferation (1–3). Overexpression of CCN3, one of the founding members of the family, inhibits proliferation in most types of tumors such as glioblastoma and Ewing sarcoma (4, 5). Similarly, down-regulation of CCN3 has been suggested to promote melanoma progression (6). On the other hand, CCN3 can also promote migration in sarcoma and glioblastoma (4, 7), although a separate study shows that it decreases the invasion of melanoma (6). Therefore, in contrast to its role in growth suppression, the role of CCN3 signaling in cell motility is less clear.Most evidence suggests CCN3 mediates its effects by binding to the integrin proteins, such as the αVβ3 receptors (8, 9), and that CCN3 alters cell adhesion in an integrin-dependent fashion (4, 10). In melanocytes, the discoidin domain receptor 1 mediates CCN3-dependent adhesion (11). CCN3 has also been observed to associate with Notch1 (12), fibulin 1C (13), S100A4 (14), and the gap junction protein Cx43³ (15, 16), suggesting that CCN3 may also modulate cell growth via non-integrin signaling pathways.Gap junction proteins are best known for forming channels between cells, contributing to intercellular communication by allowing the exchange of small ions and molecules (17, 18). Consequently, attenuated intercellular communication has been implicated in promoting carcinogenesis (19, 20). Recent evidence has indicated that connexins can mediate channel-independent growth control through interaction of their C-terminal cytoplasmic tail with various intracellular signaling molecules (21–23). In addition, many Cx43-interacting proteins, including ZO-1 (zonula occludens-1) (24), Drebrin (25), and N-cadherin (26) associate with F-actin, thus placing Cx43 in close proximity to the actin cytoskeleton.In this study, we show for the first time that CCN3 reorganizes the actin cytoskeleton of the breast cancer cells MDA-MB-231 with the formation of multiple cell protrusions, possibly by activating the small GTPase Rac1. Our results also suggest an alternative route by which Cx43 may be functionally linked to actin cytoskeletal signaling via CCN3.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Phosphorylated Tau Interacts with c-Jun N-terminal Kinase-interacting Protein 1 (JIP1) in Alzheimer Disease

In Alzheimer disease (AD) and frontotemporal dementia the microtubule-associated protein Tau becomes progressively hyperphosphorylated, eventually forming aggregates. However, how Tau dysfunction is associated with functional impairment is only partly understood, especially at early stages when Tau is mislocalized but has not yet formed aggregates. Impaired axonal transport has been proposed as a potential pathomechanism, based on cellular Tau models and Tau transgenic mice. We recently reported K369I mutant Tau transgenic K3 mice with axonal transport defects that suggested a cargo-selective impairment of kinesin-driven anterograde transport by Tau. Here, we show that kinesin motor complex formation is disturbed in the K3 mice. We show that under pathological conditions hyperphosphorylated Tau interacts with c-Jun N-terminal kinase- interacting protein 1 (JIP1), which is associated with the kinesin motor protein complex. As a result, transport of JIP1 into the axon is impaired, causing JIP1 to accumulate in the cell body. Because we found trapping of JIP1 and a pathological Tau/JIP1 interaction also in AD brain, this may have pathomechanistic implications in diseases with a Tau pathology. This is supported by JIP1 sequestration in the cell body of Tau-transfected primary neuronal cultures. The pathological Tau/JIP1 interaction requires phosphorylation of Tau, and Tau competes with the physiological binding of JIP1 to kinesin light chain. Because JIP1 is involved in regulating cargo binding to kinesin motors, our findings may, at least in part, explain how hyperphosphorylated Tau mediates impaired axonal transport in AD and frontotemporal dementia.The microtubule-associated protein Tau is predominantly found in the axonal compartment of neurons, where it binds to microtubules (1). In human brain, six isoforms of Tau are expressed, due to alternative splicing of exons 2, 3 and 10 (2). Tau consists of an amino-terminal projection domain followed by 3 or 4 microtubule binding repeats (3R or 4R), due to splicing of exon 10, and a carboxyl-terminal tail region. In the AD³ and FTD brain, Tau forms filamentous inclusions (3). They are found in nerve cell bodies and apical dendrites as neurofibrillary tangles (NFTs), in distal dendrites as neuropil threads, and in the abnormal neurites that are associated with some amyloid plaques (neuritic plaques) (3). Hyperphosphorylation of Tau is thought to be an initiating step (4), as it detaches Tau from microtubules and makes it prone to form aggregates (1, 5). Whereas in AD no mutations have been identified in the MAPT gene encoding Tau, so far 42 intronic and exonic mutations have been found in familial forms of FTD (6). Their identification assisted in the generation of transgenic mouse models that reproduce NFT formation and memory impairment (7).The models were also instrumental in testing hypotheses that had been brought forward to link Tau pathology to functional impairment (8–10). In particular, defects in axonal transport have been implicated in neurodegenerative disorders (11, 12). Tau binding to microtubules affects axonal transport (13), and in cell culture overexpression of Tau was shown to lead to impaired transport of mitochondria and vesicles (14, 15). Axonal transport defects have also been reproduced in wild-type Tau transgenic mice (16) and in K369I mutant Tau K3 mice (17), whereas Tau expression failed to inhibit axonal transport in other systems (18, 19). This apparent discrepancy may depend on the type of cargos analyzed and, specifically, the experimental paradigm, e.g. using phosphorylated (16, 17, 20) versus non-phosphorylated Tau (18).To dissect Tau-mediated axonal transport defects at a molecular level, we used K3 mice that overexpress human Tau carrying the pathogenic FTD K369I mutation (17). We observed a pronounced hyperphosphorylation of transgenic Tau in many brain areas. Clinically, the mice present with an early onset motor phenotype that is, at least in part, caused by impairment of axonal transport in neurons of the substantia nigra. Interestingly, only selected aspects of anterograde axonal transport were impaired, in particular those of kinesin-I motor complex-driven vesicles and mitochondria. Our data suggest a selective impairment of axonal transport rather than a generalized, non-selective blockage of microtubules that has been established in cell culture systems, which fail to phosphorylate Tau at the high levels that are found in vivo even under physiological conditions. More importantly, in AD and FTD Tau is even more phosphorylated, i.e. hyperphosphorylated at physiological sites and de novo at pathological sites, preventing it from binding to microtubules (1).Based on our findings of an impaired kinesin-I-driven axonal transport in the K3 mice, we speculated that hyperphosphorylated Tau may impair anterograde transport by interfering directly with components of the kinesin-I motor complex rather than disrupting the binding of the kinesin heavy chain (see below) to microtubules. Axonal transport along microtubules is mediated by members of the kinesin superfamily (KIF) of motor proteins (21–23). The KIFs typically consist of an ATPase domain that interacts with microtubules and drives movement and a domain that links to cargos, either directly or indirectly, as in the case of KIF5, by assembling with the kinesin light chain (KLC) to form the kinesin-I (KIF5/KLC) motor complex (24). In addition, increasing evidence suggests that scaffolding proteins mediate and regulate the binding of cargos to KIFs (21, 25–27). These include the scaffold protein JNK-interacting protein (JIP) that is involved in the linkage of cargos to the kinesin-I motor complex via KLC (25, 28–33).Here, by using the K3 mouse model, we identified a novel interaction of Tau and JIP in neurons that causes a trapping of JNK interacting protein 1 (JIP1) in the cell body of K3 mice, cell culture systems, and human AD brain. We found that the pathological interaction of hyperphosphorylated Tau and JIP1 competes with the physiological binding of JIP1 to KLC.     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]     

Distinct Structures of Scrapie Prion Protein (PrPSc)-seeded Versus Spontaneous Recombinant Prion Protein Fibrils Revealed by Hydrogen/Deuterium Exchange

The detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrP^Sc) are largely unknown. PrP^Sc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrP^C). One manifestation of PrP^Sc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrP^Sc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrP^Sc. Here, we compare the structures of PrP^Sc-seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ∼163–223 represents a systematically H-bonded cross-β amyloid core structure. PrP^Sc-seeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ∼145, and there is a significant degree of order within residues ∼117–133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrP^Sc-seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrP^Sc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP.Transmissible spongiform encephalopathies (TSEs),² or prion diseases, are a group of infectious neurodegenerative disorders that affect many mammalian species and include Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in cervids, and bovine spongiform encephalopathy (“mad cow” disease) (1–7). All of these diseases appear to be intimately associated with conformational conversion of the normal host-encoded prion protein, termed PrP^C, to a pathological isoform, PrP^Sc (1–5). According to the “protein-only” model, PrP^Sc itself represents the infectious prion agent (1, 8); it is believed to self-propagate by an autocatalytic mechanism involving binding to PrP^C and templating the conversion of the latter protein to the PrP^Sc state (9, 10). Although molecular details of such a mechanism of disease propagation remain largely unknown, the general principle of protein-based infectivity is supported by a wealth of experimental data (1–7).PrP^C is a monomeric glycophosphatidylinositol-linked glycoprotein that is highly protease-sensitive and soluble in nonionic detergents. High resolution NMR data show that the recombinant PrP (rPrP), a nonglycosylated model of PrP^C, consists of a flexible N-terminal region and a folded C-terminal domain encompassing three α-helices and two short β-strands (11–13). Conversely, the PrP^Sc isoform is aggregate in nature, rich in β-sheet structure, insoluble in nonionic detergents, and partially resistant to proteinase K (PK) digestion, with a PK-resistant core encompassing the C-terminal ∼140 residues (1–5, 14, 15). Little specific structural information is available, however, for this isoform beyond low resolution biochemical and spectroscopic characterization. Thus, the structure of PrP^Sc conformer(s) associated with prion infectivity remains one of the best guarded mysteries, hindering efforts to understand the molecular basis of TSE diseases.Many efforts have been made over the years to recapitulate PrP^Sc formation and prion propagation in vitro. Early studies have shown that PrP^C can be converted with remarkable species and strain specificities to a PrP^Sc-like conformation (as judged by PK resistance) simply by incubation with PrP^Sc from prion-infected animals (16, 17). The yields of these original cell-free conversion experiments were low, and no new infectivity could be attributed to the newly converted material (18). An important more recent study showed that both PrP^Sc and TSE infectivity can be amplified indefinitely in crude brain homogenates using successive rounds of sonication and incubation (19), a procedure called protein misfolding cyclic amplification (PMCA) (20). Similar amplification of the TSE infectivity was also accomplished by PMCA employing purified PrP^C as a substrate, although only in the presence of polyanions such as RNA and copurified lipids (21). Unfortunately, the quantities of infectious PrP^Sc generated by PMCA using purified brain-derived PrP^C are very small, precluding most structural studies.In contrast to brain-derived PrP^C, large scale purification can be readily accomplished for bacterially expressed rPrP, a form of PrP lacking glycosylation and the glycophosphatidylinositol anchor. The latter protein can spontaneously polymerize into amyloid fibrils, and much insight has been gained into mechanistic and structural aspects of this reaction (22–28). However, although rPrP fibrils were shown to cause or accelerate a transmissible neurodegenerative disorder in transgenic mice overexpressing a PrP^C variant encompassing residues 89–231, the infectivity titer of these “synthetic prions” was extremely low (29) or absent altogether (4). This low infectivity coincides with much shorter PK-resistant core of rPrP amyloid fibrils compared with brain-derived PrP^Sc (26, 30), raising questions regarding the relationship between these fibrils and the authentic TSE agent. In this context, an important recent development was the finding that the PrP^Sc-seeded PMCA method can be extended to rPrP, yielding protease-resistant recombinant PrP aggregates (rPrP^PMCA or rPrP-res^(Sc)) (31). These aggregates display a PK digestion pattern that is much more closely related to PrP^Sc than that of previously studied spontaneously formed rPrP fibrils, offering a potentially more relevant model for biochemical and biophysical studies. Here, we provide, for the first time, a direct insight into the structure of rPrP^PMCA. H/D exchange data coupled with MS analysis (HXMS) allowed us to identify systematically H-bonded core region(s) of these aggregates, shedding a new light on the mechanisms underlying formation of PK-resistant structures.     

Integrative Proteomic Profiling of Protein Activity and Interactions Using Protein Arrays

Proteomic studies based on abundance, activity, or interactions have been used to investigate protein functions in normal and pathological processes, but their combinatory approach has not been attempted. We present an integrative proteomic profiling method to measure protein activity and interaction using fluorescence-based protein arrays. We used an on-chip assay to simultaneously monitor the transamidating activity and binding affinity of transglutaminase 2 (TG2) for 16 TG2-related proteins. The results of this assay were compared with confidential scores provided by the STRING database to analyze the functional interactions of TG2 with these proteins. We further created a quantitative activity-interaction map of TG2 with these 16 proteins, categorizing them into seven groups based upon TG2 activity and interaction. This integrative proteomic profiling method can be applied to quantitative validation of previously known protein interactions, and in understanding the functions and regulation of target proteins in biological processes of interest.Proteomics is the large-scale analysis of whole proteins and their role in biological systems. Abundance-based proteomics assigns protein functions in normal and pathological processes by quantification of global differences in protein expression levels (1). This classic approach identifies functional biomarkers by comparing samples from healthy individuals and patients. However, this abundance-based approach provides only indirect information about protein function (2). The abundance of a protein is not necessarily correlated with its activity because protein activities are predominantly regulated by a series of post-translational modifications (1, 2). Activity-based proteomics (activity-based protein profiling) is therefore considered an alternative approach to assigning protein functions in biological processes of interest (3). In this approach, specific activity-based probes using fluorescent, radioactive, and affinity tags are usually designed for detection of protein activity (2, 4–6). Activity-based proteomics identifies markers by comparative analyses of activity profiles between healthy and diseased cells and tissues (3, 7, 8). This approach is also used for profiling enzyme inhibitors, for developing therapeutic reagents, and for diagnosis (2, 5). Another functional proteomic approach using large-scale analysis is interactomics or interaction proteomics, which is a useful method for understanding the regulation of proteins in biological systems (9). To elucidate bioactive protein interactions with proteins or ligands, a number of technologies are currently used including the yeast two-hybrid system, affinity purification and mass spectrometry, the protein fragment complementation assay, the luminescence-based mammalian interactome, and protein arrays (9–13). Global differences in the dynamics of the interactome between healthy and diseased individuals provide new insights into causes of disease and can be used for biomarker identification and drug discovery (14–16). Thus, combinatory analyses of abundance, activity, and interaction have great potential in revealing regulation mechanisms and functions of proteins, although such an integrated proteomic approach has not been widely used.These proteomic methods have been coupled with various detection methods including one- or two-dimensional gel electrophoresis, one- or two-dimensional liquid chromatography and tandem mass spectrometry, surface plasmon resonance, and fluorometric assays for analyses of the proteome (9, 11). In combination with specific probes, colorimetric and fluorometric assays using multiwell plates have been extensively used for the determination of the abundance and activity of various proteins. Although often limited by the amount of sample, these methods nonetheless facilitate real-time measurement of changes in protein activity and high-throughput analyses of protein abundances and activities (17). Surface plasmon resonance, a method that does not necessitate labeling of proteins, has also been used for analysis of protein abundance, activity, and binding affinity (18–20). Using only very small amounts of sample, the microarray combined with fluorometric probes is a promising technology for the rapid analysis of a wide variety of biomolecular interactions, protein abundances, and activities. This approach has been used for serodiagnosis and identification of biomarkers by abundance-based protein profiling in human sera (21–25). It has also been used for kinetic studies of carbohydrate-protein (17) and peptide-protein interactions (26, 27). In addition, this technology has been used for the rapid determination of enzyme activities and for the identification of enzyme substrates and inhibitors (24, 28–33). However, combinatory profiling of protein activities and interactions based on array technology has yet to be reported.Using protein arrays, we propose as a model system an integrative proteomic approach for simultaneous profiling of the transamidating activity and interactions of transglutaminase 2 (TG2) with TG2-related proteins. TG2, known as tissue transglutaminase, is a member of the calcium-dependent transglutaminase family. Its activity and interactions are associated with a wide variety of diseases and cellular events (34). TG2 is implicated in the pathogenesis of a wide variety of diseases including inflammatory diseases such as celiac sprue, neurodegenerative disorders such as Huntington''s, Alzheimer''s, and Parkinson''s disease, as well as cancers, cardiovascular diseases, and diabetes (34–36). TG2 is also involved in various cellular events including cell growth, cell differentiation, cell adhesion, extracellular matrix crosslinking, and apoptosis (34, 37, 38). In the present study, the transamidating activity and binding affinity of TG2 for 16 proteins were simultaneously monitored using Cy5-conjugated TG2 and protein arrays (Fig. 1). Using this large-scale analysis, we constructed a quantitative activity-interaction (AI)¹ map to describe the quantitative interaction of TG2 with its related proteins. Thus, this integrative proteomic approach can be used to characterize functions and regulation mechanisms of a target protein in many biological processes of interest.Open in a separate windowFig. 1.Schematic diagram for the simultaneous analysis of transamidating activity and interaction of TG2 with TG2-related proteins. BAPA, 5-(biotinamido)pentylamine; Pr, protein; SA, streptavidin; TG2, transglutaminase 2.     

Identification of Tumor-associated Autoantigens for the Diagnosis of Colorectal Cancer in Serum Using High Density Protein Microarrays

There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers.Colorectal cancer (CRC)¹ is the second most prevalent cancer in the western world. The development of this disease takes decades and involves multiple genetic events. CRC remains a major cause of mortality in developed countries because most of the patients are diagnosed at advanced stages because of the reluctance to use highly invasive diagnostic tools like colonoscopy. Actually only a few proteins have been described as biomarkers in CRC (carcinoembryonic antigen (CEA), CA19.9, and CA125 (1–3)), although none of them is recommended for clinical screening (4). Proteomics analysis is actively used for the identification of new biomarkers. In previous studies, the use of two-dimensional DIGE and antibody microarrays allowed the identification of differentially expressed proteins in CRC tissue, including isoforms and post-translational modifications responsible for modifications in signaling pathways (5–8). Both approaches resulted in the identification of a collection of potential tumoral tissue biomarkers that is currently being investigated.However, the implementation of simpler, non-invasive methods for the early detection of CRC should be based on the identification of proteins or antibodies in serum or plasma (9–13). There is ample evidence of the existence of an immune response to cancer in humans as demonstrated by the presence of autoantibodies in cancer sera. Self-proteins (autoantigens) altered before or during tumor formation can elicit an immune response (13–19). These tumor-specific autoantibodies can be detected at early cancer stages and prior to cancer diagnosis revealing a great potential as biomarkers (14, 15, 20). Tumor proteins can be affected by specific point mutations, misfolding, overexpression, aberrant glycosylation, truncation, or aberrant degradation (e.g. p53, HER2, NY-ESO1, or MUC1 (16, 21–25)). In fact, a number of tumor-associated autoantigens (TAAs) were identified previously in different studies involving autoantibody screening in CRC (26–28).Several approaches have been used to identify TAAs in cancer, including natural protein arrays prepared with fractions obtained from two-dimensional LC separations of tumoral samples (29, 30) or protein extracts from cancer cells or tissue (9, 31) probed by Western blot with patient sera, cancer tissue peptide libraries expressed as cDNA expression libraries for serological screening (serological analysis of recombinant cDNA expression libraries (SEREX)) (22, 32), or peptides expressed on the surface of phages in combination with microarrays (17, 18, 33, 34). However, these approaches suffer from several drawbacks. In some cases TAAs have to be isolated and identified from the reactive protein lysate by LC-MS techniques, or in the phage display approach, the reactive TAA could be a mimotope without a corresponding linear amino acid sequence. Moreover, cDNA libraries might not be representative of the protein expression levels in tumors as there is a poor correspondence between mRNA and protein levels.Protein arrays provide a novel platform for the identification of both autoantibodies and their respective TAAs for diagnostic purposes in cancer serum patients. They present some advantages. Proteins printed on the microarray are known “a priori,” avoiding the need for later identifications and the discovery of mimotopes. There is no bias in protein selection as the proteins are printed at a similar concentration. This should result in a higher sensitivity for biomarker identification (13, 35, 36).In this study, we used commercially available high density protein microarrays for the identification of autoantibody signatures and tumor-associated antigens in colorectal cancer. We screened 20 CRC patient and control sera with protein microarrays containing 8000 human proteins to identify the CRC-associated autoantibody repertoire and the corresponding TAAs. Autoantibody profiles that discriminated the different types of CRC metastasis were identified. Moreover a set of TAAs showing increased or decreased expression in cancer cell lines and paired tumoral tissues was found. Finally an ELISA was set up to test the ability of the most immunoreactive proteins to detect colorectal adenocarcinoma. On the basis of the antibody response, combinations of three antigens, PIM1, MAPKAPK3, and ACVR2B, showed a great potential for diagnosis.     

Equivalence of Protein Inventories Obtained from Formalin-fixed Paraffin-embedded and Frozen Tissue in Multidimensional Liquid Chromatography-Tandem Mass Spectrometry Shotgun Proteomic Analysis

Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for retrospective biomarker discovery studies, and recent work indicates the feasibility of using shotgun proteomics to characterize FFPE tissue proteins. A critical question in the field is whether proteomes characterized in FFPE specimens are equivalent to proteomes in corresponding fresh or frozen tissue specimens. Here we compared shotgun proteomic analyses of frozen and FFPE specimens prepared from the same colon adenoma tissues. Following deparaffinization, rehydration, and tryptic digestion under mild conditions, FFPE specimens corresponding to 200 μg of protein yielded ∼400 confident protein identifications in a one-dimensional reverse phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The major difference between frozen and FFPE proteomes was a decrease in the proportions of lysine C-terminal to arginine C-terminal peptides observed, but these differences had little effect on the proteins identified. No covalent peptide modifications attributable to formaldehyde chemistry were detected by analyses of the MS/MS datasets, which suggests that undetected, cross-linked peptides comprise the major class of modifications in FFPE tissues. Fixation of tissue for up to 2 days in neutral buffered formalin did not adversely impact protein identifications. Analysis of archival colon adenoma FFPE specimens indicated equivalent numbers of MS/MS spectral counts and protein group identifications from specimens stored for 1, 3, 5, and 10 years. Combination of peptide isoelectric focusing-based separation with reverse phase LC-MS/MS identified 2554 protein groups in 600 ng of protein from frozen tissue and 2302 protein groups from FFPE tissue with at least two distinct peptide identifications per protein. Analysis of the combined frozen and FFPE data showed a 92% overlap in the protein groups identified. Comparison of gene ontology categories of identified proteins revealed no bias in protein identification based on subcellular localization. Although the status of posttranslational modifications was not examined in this study, archival samples displayed a modest increase in methionine oxidation, from ∼17% after one year of storage to ∼25% after 10 years. These data demonstrate the equivalence of proteome inventories obtained from FFPE and frozen tissue specimens and provide support for retrospective proteomic analysis of FFPE tissues for biomarker discovery.Formalin-fixed paraffin-embedded (FFPE)¹ tissue samples are routinely prepared during the pathological characterization of clinical specimens and are abundantly available in pathology archives worldwide. The fixation process yields clinically relevant samples that can be stored at ambient temperature and are suitable for pathological examination by light microscopy even after years in storage. Given the wealth of clinical data associated with specimens collected over a span of decades, such as patient treatment regimens and outcomes, FFPE tissue represents a potentially valuable resource for biomarker discovery through retrospective analysis (1, 2).However, fixation of tissue in formalin leads to significant cross-linking among proteins and other biomolecules, rendering the samples incompatible with many biochemical analyses. Immunohistochemical (IHC) analysis of FFPE tissue has been conducted since the 1970s using either proteolysis or protein denaturants to expose antigenic regions of proteins (3, 4). Since the 1990s, detection of antigens in FFPE tissue has been improved through the development of so-called antigen retrieval techniques (5, 6). These methods involve application of heat in the presence of any of a variety of buffers resulting in the cleavage of methylene bridges formed during the course of fixation (2).Despite their utilization for IHC analysis, FFPE tissue samples have been largely overlooked in proteomics studies, due to the assumption that tissue fixation would make proteomic analysis intractable. Recent work appears to refute this notion. In 2005, Hood et al. (7) first described the successful application of shotgun proteome analysis to FFPE tissue. Using laser capture microdissected cells and an optimized extraction method, hundreds of proteins were identified from a cancerous prostate lesion and benign prostate hyperplasia, thus opening the door to comparative proteomic analyses of FFPE tissue. Moreover, the same study showed that the numbers and identities of proteins observed were remarkably similar when applying the method to frozen and FFPE mouse liver, thus lending support to the use of FFPE tissue in biomarker discovery studies. Since the initial demonstration of its feasibility, FFPE tissues from diverse origins including breast, liver, kidney, lymphoma, and bone successfully have been subjected to proteomic analyses (8–14).Although this work suggests the feasibility of biomarker discovery from FFPE tissue, most of these previous studies have been performed on small amounts of material with one-dimensional reverse phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. The use of multidimensional peptide separations can extend the dynamic range of the LC-MS/MS analyses to detect lower abundance proteins. Recently, the use of capillary isotachophoresis as the first dimension in a multidimensional peptide separation strategy for analyzing FFPE tissue was described (8). In this study, thousands of proteins were identified out of <4 μg of digest from FFPE human liver sections. However, the apparatus used was an in-house, custom-designed system, not readily accessible to other laboratories. In several of these studies, proteins identified by a single peptide were accepted as valid identifications. Use of single peptide-based identifications elevates the probability of false positive protein identifications, and these identifications often constitute the majority of protein identifications (15).The equivalence of fresh/frozen and FFPE tissue proteomes is a critical issue in evaluating the suitability of employing FFPE tissues for biomarker discovery by comparative proteomic analyses. Hood et al. (7) and Guo et al. (14) reported comparisons from analyses of paired fresh and frozen tissue specimens. Guo et al. (14) reported an apparent overlap of 83% in protein identifications between FFPE and frozen brain tissue specimens, whereas Hood et al. (7) did not report the degree of overlap, but found that FFPE mouse liver tissue yielded about 88% of the identifications determined for frozen mouse liver tissue. The majority of protein identifications in both studies were based on single peptide assignments. These investigations did not explicitly address the effect of formaldehyde-derived modifications on the inventories of identified peptides.An unexplored question with FFPE tissue specimens is the extent to which normal variability in fixation process and storage duration affect the proteomes observed. The duration of tissue fixation is not highly standardized and may vary from hours to several days. One of the most attractive features of FFPE specimens is the opportunity for retrospective biomarker discovery, but the effects of storage for many years on tissue proteomes remains unknown.Here, we address these questions through detailed comparative studies of the analysis of fresh frozen and FFPE tissues by LC-MS/MS-based shotgun proteomics. We used the same fresh tissue specimens to prepare both frozen and FFPE samples for paired comparisons. We evaluated conditions for tissue lysis and digestion and the effects of fixation time and storage duration on the number of protein IDs obtained during shotgun proteomic analysis of FFPE tissue. We also characterized the differences in peptides observed between fixed and frozen specimens in an effort to understand the effect of fixation from a practical biomarker discovery standpoint. Furthermore, we compared analyses of fresh frozen and FFPE colon adenoma tissue by multidimensional LC-MS/MS using gel-based isoelectric focusing of peptides (Fig. 1). The results demonstrate a remarkable overlap in the number and identities of proteins between the fixed and frozen tissue and indicate that variations in duration of fixation and storage have a minimal effect on protein inventories obtained by shotgun proteomic analysis. The data indicate essential equivalence between protein inventories obtained from fresh frozen and FFPE tissue specimens by shotgun proteomics and validate the use of FFPE tissue specimens for biomarker discovery.Open in a separate windowFig. 1.Strategy for multidimensional LC-MS/MS analysis of FFPE tissue.