Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)

Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues.Many human diseases are characterized by abnormalities in complex signaling pathways (1). The expression and activation status of proteins from these deregulated pathways has traditionally been analyzed using single marker techniques such as immunohistochemistry and Western blotting. Although these techniques have provided valuable information on the molecular abnormalities underlying human disease, they are labor intensive, have a low throughput, and often require high sample volume. Furthermore, techniques such as Western blotting are not applicable in the routine clinical setting. Miniaturized parallel immunoassay techniques have been developed in recent years and have played a pivotal role in biomarker discovery (2). Antibody arrays enable multiple potential disease markers to be investigated in a single sample in parallel (3). Beyond this, Reverse Phase Protein Arrays (RPPA)¹ are sensitive high throughput tools that can quantify protein expression levels and activation status (posttranslational modifications such as phosphorylation) in multiple experimental samples simultaneously. The technique requires only minute amounts of samples, printed as lysate arrays onto slides, and hundreds of markers of interest can be investigated, array by array, in a miniaturized dot blot manner. Numerous reports have demonstrated that RPPA can be applied to various sources of cells and tissues to analyze protein profiles, signaling pathway networks, and for the identification of biomarkers (4–13). A recently published workshop report reviews the full potential and advances of RPPA for use in clinical, translational, and basic research (11).In oncology, the parallel profiling of multiple protein markers is particularly desirable to study tumor initiation and progression, to classify tumor disease states on the molecular level, and to discover and monitor biomarkers that can predict therapeutic response or tumor recurrence (14–16). The study of signaling response and analysis of pharmacodynamic (PD) markers upon treatment using in vitro and in vivo test systems (e.g. cell line or patient derived xenograft tumor models) is an established component of preclinical and early clinical drug development. These techniques can provide evidence of target pathway modulation for new therapeutic lead candidate compounds and provide valuable information on the drug mode of action (17), especially in the translational phase. Multiplex analyses of PD biomarkers by RPPA have been performed in vitro using cancer cell lines (18, 19) as well as in patient-derived tumor tissue and blood samples (20, 21) to assess response to treatment and target inhibition. A combination of RPPA signaling pathway mapping and functional PET imaging has recently been successfully evaluated in xenograft models as an early response PD marker for anti-cancer drug efficacy (13).Translating miniaturized multiple protein analysis platforms-such as RPPA - from preclinical to clinical applicability is highly desirable; however, issues such as the limited amount of available clinical samples and tumor heterogeneity must first be addressed. Furthermore, most studies of RPPA in tumor tissue to date have been conducted using proteins extracted from fresh-frozen (FF) tissue specimens; whereas, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation used in clinical pathology laboratories. FFPE yields excellent tissue architecture for histological assessment and enables analysis of individual proteins in situ by techniques such as immunohistochemistry. However, formalin fixation leads to extensive protein–protein and protein–nucleic acid cross-linking (22), which can hamper protein extraction and reduce both the overall yield of extracted protein and the profile of proteins detectable by proteomic techniques (23, 24). Furthermore, formalin-induced cross-linking induces conformational changes in protein structure that can alter the immunoreactivity of some proteins in situ by hiding or altering peptide epitopes (25, 26). Such artifacts are absent from snap-frozen tissue; therefore, protein profiles obtained from FF tissue are likely to reflect the in vivo biology of the tumor more closely. However, FF tumor tissue is not widely available because it is costly to collect and maintain in the clinical setting. FFPE tissue samples are routinely archived by nearly every hospital and offer a unique opportunity to study thousands of samples retrospectively with extensive clinical records and follow-up information.Several groups have now established protocols for retrieving cross-linked proteins from fixed tissues (27–33). These methods are mainly based on the use of concentrated ionic detergents and high temperature protocols closely related to the antigen retrieval methods developed for immunohistochemistry. These studies show that obtaining nondegraded, full-length proteins from FFPE tissues for multiplex analyses is feasible (27–33). More recently, protein extraction techniques optimized for fixed samples have been used to successfully conduct RPPA using FFPE tissue biopsies from different cancer types (34–40). Guo et al. systematically investigated several protein extraction methods and demonstrated that RPPA of FFPE materials is feasible, reproducible and can generate biologically relevant protein profiles (41). Other studies have confirmed the validity of this approach and shown that data generated from RPPA analyses of FFPE tissue demonstrate good concordance with traditional immunohistochemistry markers such as HER2 protein in breast cancer (34, 40). However, to date, analyses have been performed only for a limited set of protein markers.To evaluate whether analysis of a broader panel of protein markers is feasible and generates meaningful data from FFPE tumor tissue sections, we conducted RPPA on matched samples of FF and FFPE tissues using a set of 300 markers, the largest panel reported to date. Our aim was to identify markers that performed similarly when comparing the protein profiles measured in protein extracts from matched FF and FFPE tissue, using RPPA assays established for use in frozen tissues. Correlating selected markers and assays in such a way should qualify RPPA for further use with FFPE tissues of clinical relevance, e.g. in PD marker studies. In this paper, we have specifically focused on the technical issues relevant for using the RPPA platform in a clinical setting, and did not address the biology of the test systems used in detail. However, the models used have been pre-characterized to identify key signaling parameters in context of targeted drug treatment (42). We conducted a systematic comparison of RPPA protein profiles in matched FF and FFPE tumor tissues resected from three different xenograft models of human cancer, each treated with targeted therapeutic antibodies that have previously been shown to achieve tumor growth inhibition. Furthermore, we investigated the effect of targeted drug treatment on protein expression and activation status, and the concordance of matched FF and FFPE tissue RPPA profiles. Finally, with one of the applied tumor models, we compared a set of protein profiles measured with two different multiple assay platforms - the RPPA and the Luminex Bio-Plex system, and discuss their relevance with respect to the analysis of FFPE tissue.     

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.     

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.     

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.     

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]     

Coomassie Blue as a Near-infrared Fluorescent Stain: A Systematic Comparison With Sypro Ruby for In-gel Protein Detection

Quantitative proteome analyses suggest that the well-established stain colloidal Coomassie Blue, when used as an infrared dye, may provide sensitive, post-electrophoretic in-gel protein detection that can rival even Sypro Ruby. Considering the central role of two-dimensional gel electrophoresis in top-down proteomic analyses, a more cost effective alternative such as Coomassie Blue could prove an important tool in ongoing refinements of this important analytical technique. To date, no systematic characterization of Coomassie Blue infrared fluorescence detection relative to detection with SR has been reported. Here, seven commercial Coomassie stain reagents and seven stain formulations described in the literature were systematically compared. The selectivity, threshold sensitivity, inter-protein variability, and linear-dynamic range of Coomassie Blue infrared fluorescence detection were assessed in parallel with Sypro Ruby. Notably, several of the Coomassie stain formulations provided infrared fluorescence detection sensitivity to <1 ng of protein in-gel, slightly exceeding the performance of Sypro Ruby. The linear dynamic range of Coomassie Blue infrared fluorescence detection was found to significantly exceed that of Sypro Ruby. However, in two-dimensional gel analyses, because of a blunted fluorescence response, Sypro Ruby was able to detect a few additional protein spots, amounting to 0.6% of the detected proteome. Thus, although both detection methods have their advantages and disadvantages, differences between the two appear to be small. Coomassie Blue infrared fluorescence detection is thus a viable alternative for gel-based proteomics, offering detection comparable to Sypro Ruby, and more reliable quantitative assessments, but at a fraction of the cost.Gel electrophoresis is an accessible, widely applicable and mature protein resolving technology. As the original top-down approach to proteomic analyses, among its many attributes the high resolution achievable by two dimensional gel-electrophoresis (2DE)¹ ensures that it remains an effective analytical technology despite the appearance of alternatives. However, in-gel detection remains a limiting factor for gel-based analyses; available technology generally permits the detection and quantification of only relatively abundant proteins (35). Many critical components in normal physiology and also disease may be several orders of magnitude less abundant and thus below the detection threshold of in-gel stains, or indeed most techniques. Pre- and post-fractionation technologies have been developed to address this central issue in proteomics but these are not without limitations (1–5). Thus improved detection methods for gel-based proteomics continue to be a high priority, and the literature is rich with different in-gel detection methods and innovative improvements (6–34). This history of iterative refinement presents a wealth of choices when selecting a detection strategy for a gel-based proteomic analysis (35).Perhaps the best known in-gel detection method is the ubiquitous Coomassie Blue (CB) stain; CB has served as a gel stain and protein quantification reagent for over 40 years. Though affordable, robust, easy to use, and compatible with mass spectrometry (MS), CB staining is relatively insensitive. In traditional organic solvent formulations, CB detects ∼ 10 ng of protein in-gel, and some reports suggest poorer sensitivity (27, 29, 36, 37). Sensitivity is hampered by relatively high background staining because of nonspecific retention of dye within the gel matrix (32, 36, 38, 39). The development of colloidal CB (CCB) formulations largely addressed these limitations (12); the concentration of soluble CB was carefully controlled by sequestering the majority of the dye into colloidal particles, mediated by pH, solvent, and the ionic strength of the solution. Minimizing soluble dye concentration and penetration of the gel matrix mitigated background staining, and the introduction of phosphoric acid into the staining reagent enhanced dye-protein interactions (8, 12, 40), contributing to an in-gel staining sensitivity of 5–10 ng protein, with some formulations reportedly yielding sensitivities of 0.1–1 ng (8, 12, 22, 39, 41, 42). Thus CCB achieved higher sensitivity than traditional CB staining, yet maintained all the advantages of the latter, including low cost and compatibility with existing densitometric detection instruments and MS. Although surpassed by newer methods, the practical advantages of CCB ensure that it remains one of the most common gel stains in use.Fluorescent stains have become the routine and sensitive alternative to visible dyes. Among these, the ruthenium-organometallic family of dyes have been widely applied and the most commercially well-known is Sypro Ruby (SR), which is purported to interact noncovalently with primary amines in proteins (15, 18, 19, 43). Chief among the attributes of these dyes is their high sensitivity. In-gel detection limits of < 1 ng for some proteins have been reported for SR (6, 9, 14, 44, 45). Moreover, SR staining has been reported to yield a greater linear dynamic range (LDR), and reduced interprotein variability (IPV) compared with CCB and silver stains (15, 19, 46–49). SR is easy to use, fully MS compatible, and relatively forgiving of variations in initial conditions (6, 15). The chief consequence of these advances remains high cost; SR and related stains are notoriously expensive, and beyond the budget of many laboratories. Furthermore, despite some small cost advantage relative to SR, none of the available alternatives has been consistently and quantitatively demonstrated to substantially improve on the performance of SR under practical conditions (9, 50).Notably, there is evidence to suggest that CCB staining is not fundamentally insensitive, but rather that its sensitivity has been limited by traditional densitometric detection (50, 51). When excited in the near IR at ∼650 nm, protein-bound CB in-gel emits light in the range of 700–800 nm. Until recently, the lack of low-cost, widely available and sufficiently sensitive infrared (IR)-capable imaging instruments prevented mainstream adoption of in-gel CB infrared fluorescence detection (IRFD); advances in imaging technology are now making such instruments far more accessible. Initial reports suggested that IRFD of CB-stained gels provided greater sensitivity than traditional densitometric detection (50, 51). Using CB R250, in-gel IRFD was reported to detect as little as 2 ng of protein in-gel, with a LDR of about an order of magnitude (2 to 20 ng, or 10 to 100 ng in separate gels), beyond which the fluorescent response saturated into the μg range (51). Using the G250 dye variant, it was determined that CB-IRFD of 2D gels detected ∼3 times as many proteins as densitometric imaging, and a comparable number of proteins as seen by SR (50). This study also concluded that CB-IRFD yielded a significantly higher signal to background ratio (S/BG) than SR, providing initial evidence that CB-IRFD may be superior to SR in some aspects of stain performance (50).Despite this initial evidence of the viability of CB-IRF as an in-gel protein detection method, a detailed characterization of this technology has not yet been reported. Here a more thorough, quantitative characterization of CB-IRFD is described, establishing its lowest limit of detection (LLD), IPV, and LDR in comparison to SR. Finally a wealth of modifications and enhancements of CCB formulations have been reported (8, 12, 21, 24, 26, 29, 40, 41, 52–54), and likewise there are many commercially available CCB stain formulations. To date, none of these formulations have been compared quantitatively in terms of their relative performance when detected using IRF. As a general detection method for gel-based proteomics, CB-IRFD was found to provide comparable or even slightly superior performance to SR according to most criteria, including sensitivity and selectivity (50). Furthermore, in terms of LDR, CB-IRFD showed distinct advantages over SR. However, assessing proteomes resolved by 2DE revealed critical distinctions between CB-IRFD and SR in terms of protein quantification versus threshold detection: neither stain could be considered unequivocally superior to the other by all criteria. Nonetheless, IRFD proved the most sensitive method of detecting CB-stained protein in-gel, enabling high sensitivity detection without the need for expensive reagents or even commercial formulations. Overall, CB-IRFD is a viable alternative to SR and other mainstream fluorescent stains, mitigating the high cost of large-scale gel-based proteomic analyses, making high sensitivity gel-based proteomics accessible to all labs. With improvements to CB formulations and/or image acquisition instruments, the performance of this detection technology may be further enhanced.     

ASAP1, a Phospholipid-Dependent Arf GTPase-Activating Protein That Associates with and Is Phosphorylated by Src

Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4,5-biphosphate (PIP₂)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP₂-dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization, ASAP1 could coordinate membrane remodeling events with these processes.Membrane traffic, the transfer of material between membrane-bound compartments, is needed for such diverse cellular processes as secretion, endocytosis, and changes in cell shape that accompany cell growth, division, and migration (reviewed in references 84, 85, and 87). It is mediated by transport vesicles that are formed by budding from a donor membrane. The process of budding is driven by the assembly of a proteinaceous coat. Once the vesicle is formed, the coat must dissociate to permit fusion with an acceptor membrane and the consequent delivery of the vesicle’s contents. These steps are regulated in part by the Arf family of small GTP-binding proteins (reviewed in references 8, 23, 61, and 63). Arfs are highly conserved and are found in eukaryotes ranging from yeast to humans. The mammalian Arf family is divided into several classes based largely on sequence similarity: class I (Arfs 1 through 3), class II (Arfs 4 and 5), class III (Arf6), and the more distantly related Arf-like (Arl) class. By linking GTP binding and hydrolysis to coat assembly and disassembly, Arfs regulate membrane trafficking at a number of sites. Arf1 has been implicated in endoplasmic reticulum-to-Golgi and intra-Golgi transport, endosome-to-endosome fusion, and synaptic vesicle formation (8, 23, 28, 61, 63, 66). Arf6 has been implicated in regulation of membrane traffic between the plasma membrane and a specialized endocytic compartment, and its function has been linked to cytoskeletal reorganization (25, 26, 71, 73, 74). The specific sites of action of the other Arf family members are not known.The hydrolysis of GTP on Arf requires a GTPase-activating protein (GAP) (19, 61). With multiple Arfs and multiple sites of action, the existence of several unique Arf GAPs had been anticipated. A number of activities have been purified or partially purified from mammalian sources, including rat liver (19, 57, 77), rat spleen (21), and bovine brain (79), and two Arf GAP activities from rat liver have been resolved (77). They have similar Arf specificities but differ in their lipid dependencies. One of the Arf GAPs (ArfGAP/ArfGAP1, hereafter referred to as ArfGAP1) which functions in the Golgi is activated by dioleoglycerols (3, 4, 19, 40). ArfGAP1, in common with a yeast Arf GAP, GCS1 (72), contains a zinc finger domain which is required for activity (19). The second Arf GAP (ArfGAP2) is specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatidic acid (PA). Based on lipid requirements, ArfGAP2 was speculated to function at the plasma membrane and be regulated independently of ArfGAP1 (77). ArfGAP1 and ArfGAP2 were antigenically distinct and, therefore, likely to be distinct gene products; however, prior to this study, only ArfGAP1 had been cloned (19).Src, a cytoplasmic tyrosine kinase with N-terminal Src homology 3 (SH3) and SH2 domains, transduces signals important for cell growth and cytoskeletal organization (12, 68, 91). A number of studies suggest that Src is also involved in regulating membrane traffic. Src associates primarily with endosomal membranes and in several cell types has been localized to specialized secretory vesicles, including synaptic vesicles (5, 20, 34, 46, 54, 69, 81). Overexpression of Src accelerates endocytosis (95). In addition, Src associates with or phosphorylates several proteins involved in membrane trafficking (5, 31, 43, 65).Here, we report the purification and cloning of a PIP₂-dependent Arf GAP, ASAP1. ASAP1 contains a zinc finger domain similar to that required for GAP activity in ArfGAP1 and GCS1. ASAP1 also contains a number of domains that are likely to be involved in regulation and/or localization: a pleckstrin homology (PH) domain, three ankyrin (ANK) repeats, a proline-rich region with SH3 binding motifs, and an SH3 domain. In addition, ASAP1 was identified independently as a binding protein for Src and was found to be phosphorylated on tyrosine in cells that express activated Src. ASAP1 also associated with the adapter protein c-Crk in vitro. ASAP1 was localized to the cytoplasm and the cell edge likely associated with the plasma membrane. We propose that ASAP1, by binding both Src and PIP₂, could coordinate membrane trafficking with cell growth or actin cytoskeleton remodeling.     

Plasma Protein Pentosidine and Carboxymethyllysine, Biomarkers for Age-related Macular Degeneration

Age-related macular degeneration (AMD) causes severe vision loss in the elderly; early identification of AMD risk could help slow or prevent disease progression. Toward the discovery of AMD biomarkers, we quantified plasma protein N^ε-carboxymethyllysine (CML) and pentosidine from 58 AMD and 32 control donors. CML and pentosidine are advanced glycation end products that are abundant in Bruch membrane, the extracellular matrix separating the retinal pigment epithelium from the blood-bearing choriocapillaris. We measured CML and pentosidine by LC-MS/MS and LC-fluorometry, respectively, and found higher mean levels of CML (∼54%) and pentosidine (∼64%) in AMD (p < 0.0001) relative to normal controls. Plasma protein fructosyl-lysine, a marker of early glycation, was found by amino acid analysis to be in equal amounts in control and non-diabetic AMD donors, supporting an association between AMD and increased levels of CML and pentosidine independent of other diseases like diabetes. Carboxyethylpyrrole (CEP), an oxidative modification from docosahexaenoate-containing lipids and also abundant in AMD Bruch membrane, was elevated ∼86% in the AMD cohort, but autoantibody titers to CEP, CML, and pentosidine were not significantly increased. Compellingly higher mean levels of CML and pentosidine were present in AMD plasma protein over a broad age range. Receiver operating curves indicate that CML, CEP adducts, and pentosidine alone discriminated between AMD and control subjects with 78, 79, and 88% accuracy, respectively, whereas CML in combination with pentosidine provided ∼89% accuracy, and CEP plus pentosidine provided ∼92% accuracy. Pentosidine levels appeared slightly altered in AMD patients with hypertension and cardiovascular disease, indicating further studies are warranted. Overall this study supports the potential utility of plasma protein CML and pentosidine as biomarkers for assessing AMD risk and susceptibility, particularly in combination with CEP adducts and with concurrent analyses of fructosyl-lysine to detect confounding factors.Age-related macular degeneration (AMD)¹ is a progressive, multifactorial disease and a major cause of severe vision loss in the elderly (1). Deposition of debris (drusen) in the macular region of Bruch membrane, the extracellular matrix separating the choriocapillaris from the retinal pigment epithelium (RPE), is an early, hallmark risk factor of AMD. The disease can progress to advanced dry AMD (geographic atrophy), which is characterized by regional degeneration of photoreceptor and RPE cells, or to advanced wet AMD (choroidal neovascularization (CNV)), which is characterized by abnormal blood vessels growing from the choriocapillaris through Bruch membrane beneath the retina. CNV accounts for over 80% of debilitating vision loss in AMD; however, only 10–15% of AMD cases progress to CNV.There is growing consensus that AMD is an age-related inflammatory disease involving dysregulation of the complement system; however, triggers of the inflammatory response have yet to be well defined. Oxidative stress appears to be involved as smoking significantly increases the risk of AMD (2), antioxidant vitamins can selectively slow AMD progression (3), and a host of oxidative protein and DNA modifications have been detected at elevated levels in AMD Bruch membrane, drusen, retina, RPE, and plasma (4–11). Oxidative protein modifications like carboxyethylpyrrole (CEP) and N^ε-carboxymethyllysine (CML), both elevated in AMD Bruch membrane, stimulate neovascularization in vivo (12, 13), suggesting possible roles in CNV. Other studies have shown that mice immunized with CEP protein modifications develop an AMD-like phenotype (14). Accordingly oxidative modifications may be catalysts or triggers of AMD pathology (6).AMD has long been hypothesized to be a systemic disease (15) based in part on the presence of retinal drusen in patients with membranoproliferative glomerulonephritis type II (16) and systemic complement activation in AMD (17). Support for this hypothesis also comes from mounting evidence that advanced glycation end products (AGEs) may play a role in AMD (4, 5, 7, 18, 19). AGEs are a heterogeneous group of mostly oxidative modifications resulting from the Maillard nonenzymatic glycation reaction that have been associated with age-related diseases and diabetic complications (20, 21). In 1998, CML was the first AGE to be found in AMD Bruch membrane and drusen (4). Other AGEs have since been detected in AMD ocular tissues (5, 7, 18) and in Bruch membrane, drusen, RPE, and choroidal extracellular matrix from healthy eyes (6, 22). CML, a nonfluorescent AGE, and pentosidine, a fluorescent cross-linking AGE, increase with age in Bruch membrane (18, 23). Receptors for AGEs (RAGE and AGE-R1) appear elevated on RPE and photoreceptor cells in early and advanced dry AMD (7) especially in RPE overlying drusen-like deposits on Bruch membrane (19). AGE-R3, also known as galectin-3, is elevated in AMD Bruch membrane (24).Although AMD susceptibility genes now account for over 50% of AMD cases (25), many individuals with AMD risk genotypes may never develop advanced disease with severe vision loss. Nevertheless the prevalence of advanced AMD is increasing (26). Toward the discovery of better methods to detect those at risk for advanced AMD, we quantified CML and pentosidine in plasma proteins from AMD and control patients and compared their discriminatory accuracy with plasma CEP biomarkers. CEP biomarkers have been shown to enhance the AMD predictive accuracy of genomic AMD biomarkers (11). This report shows CML and pentosidine to be elevated in AMD plasma proteins and demonstrates their potential biomarker utility in assessing AMD risk and susceptibility especially in combination with CEP biomarkers.