RESUMO
Recently, a novel technology was published, utilizing the strengths of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) and immunohistochemistry (IHC), achieving highly multiplexed, targeted imaging of biomolecules in tissue. This new technique, called MALDI-IHC, opened up workflows to target molecules of interest using MALDI-MSI that are usually targeted by standard IHC. In this paper, the utility of targeted MALDI-IHC and its complementarity with untargeted on-tissue bottom-up spatial proteomics is explored using breast cancer tissue. Furthermore, the MALDI-2 effect was investigated and demonstrated to improve MALDI-IHC. Formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections were stained for multiplex MALDI-IHC with six photocleavable mass-tagged (PC-MT) antibodies constituting a breast cancer antibody panel (CD20, actin-αSM, HER2, CD68, vimentin, and panCK). K-means spatial clusters were created based on the MALDI-IHC images and cut out using laser-capture microdissection (LMD) for further untargeted LC-MS-based bottom-up proteomics analyses. Numerous peptides could be tentatively assigned to multiple proteins, of which three proteins were also part of the antibody panel (vimentin, keratins, and actin). Post-ionization with MALDI-2 showed an increased intensity of the PC-MTs and suggests options for the development of new mass-tags. Although the on-tissue digestion covered a wider range of proteins, the MALDI-IHC allowed for easy and straightforward identification of proteins that were not detected in untargeted approaches. The combination of the multiplexed MALDI-IHC with image-guided proteomics showed great potential to further investigate diseases by providing complementary information from the same tissue section and without the need for customized instrumentation.
Assuntos
Neoplasias da Mama , Proteômica , Humanos , Feminino , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Vimentina , Proteômica/métodos , Imuno-Histoquímica , Actinas , Imagem MolecularRESUMO
The integration of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with single cell spatial omics methods allows for a comprehensive investigation of single cell spatial information and matrisomal N-glycan and extracellular matrix protein imaging. Here, the performance of the antibody-directed single cell workflows coupled with MALDI-MSI are evaluated. Miralys™ photocleavable mass-tagged antibody probes (MALDI-IHC, AmberGen, Inc.), GeoMx DSP® (NanoString, Inc.), and Imaging Mass Cytometry (IMC, Standard BioTools Inc.) were used in series with MALDI-MSI of N-glycans and extracellular matrix peptides on formalin-fixed paraffin-embedded tissues. Single cell omics protocols were performed before and after MALDI-MSI. The data suggests that for each modality combination, there is an optimal order for performing both techniques on the same tissue section. An overall conclusion is that MALDI-MSI studies may be completed on the same tissue section as used for antibody-directed single cell modalities. This work increases access to combined cellular and extracellular information within the tissue microenvironment to enhance research on the pathological origins of disease.
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Anticorpos , Polissacarídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Polissacarídeos/análise , Peptídeos/análise , Colágeno , LasersRESUMO
A recently discovered natural family of light-gated anion channelrhodopsins (ACRs) from cryptophyte algae provides an effective means of optogenetically silencing neurons. The most extensively studied ACR is from Guillardia theta (GtACR1). Earlier studies of GtACR1 have established a correlation between formation of a blue-shifted L-like intermediate and the anion channel "open" state. To study structural changes of GtACR1 in the K and L intermediates of the photocycle, a combination of low-temperature Fourier transform infrared (FTIR) and ultraviolet-visible absorption difference spectroscopy was used along with stable-isotope retinal labeling and site-directed mutagenesis. In contrast to bacteriorhodopsin (BR) and other microbial rhodopsins, which form only a stable red-shifted K intermediate at 80 K, GtACR1 forms both stable K and L-like intermediates. Evidence includes the appearance of positive ethylenic and fingerprint vibrational bands characteristic of the L intermediate as well as a positive visible absorption band near 485 nm. FTIR difference bands in the carboxylic acid CâO stretching region indicate that several Asp/Glu residues undergo hydrogen bonding changes at 80 K. The Glu68 â Gln and Ser97 â Glu substitutions, residues located close to the retinylidene Schiff base, altered the K:L ratio and several of the FTIR bands in the carboxylic acid region. In the case of the Ser97 â Glu substitution, a significant red-shift of the absorption wavelength of the K and L intermediates occurs. Sequence comparisons suggest that L formation in GtACR1 at 80 K is due in part to the substitution of the highly conserved Leu or Ile at position 93 in helix 3 (BR sequence) with the homologous Met105 in GtACR1.
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Temperatura Baixa , Rodopsina/química , Substituição de Aminoácidos , Ânions , Etilenos/química , Pichia/química , Conformação Proteica , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral RamanRESUMO
Optogenetics relies on the expression of specific microbial rhodopsins in the neuronal plasma membrane. Most notably, this includes channelrhodopsins, which when heterologously expressed in neurons function as light-gated cation channels. Recently, a new class of microbial rhodopsins, termed anion channel rhodopsins (ACRs), has been discovered. These proteins function as efficient light-activated channels strictly selective for anions. They exclude the flow of protons and other cations and cause hyperpolarization of the membrane potential in neurons by allowing the inward flow of chloride ions. In this study, confocal near-infrared resonance Raman spectroscopy (RRS) along with hydrogen/deuterium exchange, retinal analogue substitution, and site-directed mutagenesis were used to study the retinal structure as well as its interactions with the protein in the unphotolyzed state of an ACR from Guillardia theta (GtACR1). These measurements reveal that (i) the retinal chromophore exists as an all-trans configuration with a protonated Schiff base (PSB) very similar to that of bacteriorhodopsin (BR), (ii) the chromophore RRS spectrum is insensitive to changes in pH from 3 to 11, whereas above this pH the Schiff base (SB) is deprotonated, (iii) when Ser97, the homologue to Asp85 in BR, is replaced with a Glu, it remains in a neutral form (i.e., as a carboxylic acid) but is deprotonated at higher pH to form a blue-shifted species, (iv) Asp234, the homologue of the protonated retinylidene SB counterion Asp212 in BR, does not serve as the primary counteranion for the protonated SB, and (v) substitution of Glu68 with an Gln increases the pH at which SB deprotonation is observed. These results suggest that Glu68 and Asp234 located near the SB exist in a neutral state in unphotolyzed GtACR1 and indicate that other unidentified negative charges stabilize the protonated state of the GtACR1 SB.
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Criptófitas/química , Rodopsina/química , Substituição de Aminoácidos , Criptófitas/genética , Conformação Proteica , Retinaldeído/química , Retinaldeído/genética , Retinoides/química , Retinoides/genética , Rodopsina/genética , Bases de Schiff/química , Análise Espectral RamanRESUMO
Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 â P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs.
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Chlamydomonas reinhardtii/metabolismo , Proteínas de Plantas/metabolismo , Prótons , Rodopsina/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Rodopsina/genética , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Channelrhodopsins (ChRs) from green flagellate algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs to bioengineer their properties for specific optogenetic applications such as elucidating the function of specific neurons in brain circuits. While most studies have used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), in this work low-temperature Fourier transform infrared-difference spectroscopy is applied to study the conformational changes occurring during the primary phototransition of the red-shifted ChR1 from Chlamydomonas augustae (CaChR1). Substitution with isotope-labeled retinals or the retinal analogue A2, site-directed mutagenesis, hydrogen-deuterium exchange, and H2(18)O exchange were used to assign bands to the retinal chromophore, protein, and internal water molecules. The primary phototransition of CaChR1 at 80 K involves, in contrast to that of CrChR2, almost exclusively an all-trans to 13-cis isomerization of the retinal chromophore, as in the primary phototransition of bacteriorhodopsin (BR). In addition, significant differences are found for structural changes of the protein and internal water(s) compared to those of CrChR2, including the response of several Asp/Glu residues to retinal isomerization. A negative amide II band is identified in the retinal ethylenic stretch region of CaChR1, which reflects along with amide I bands alterations in protein backbone structure early in the photocycle. A decrease in the hydrogen bond strength of a weakly hydrogen bonded internal water is detected in both CaChR1 and CrChR2, but the bands are much broader in CrChR2, indicating a more heterogeneous environment. Mutations involving residues Glu169 and Asp299 (homologues of the Asp85 and Asp212 Schiff base counterions, respectively, in BR) lead to the conclusion that Asp299 is protonated during P1 formation and suggest that these residues interact through a strong hydrogen bond that facilitates the transfer of a proton from Glu169.
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Chlamydomonas/química , Canais Iônicos/química , Luz , Proteínas de Plantas/química , Bacteriorodopsinas/química , Bacteriorodopsinas/genética , Chlamydomonas/genética , Ligação de Hidrogênio , Canais Iônicos/genética , Isomerismo , Mutagênese Sítio-Dirigida , Processos Fotoquímicos , Proteínas de Plantas/genética , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Água/químicaRESUMO
BACKGROUND & AIMS: Using high-density human recombinant protein microarrays, we identified two potential biomarkers, kelch-like 12 (KLHL12) and hexokinase-1 (HK1), in primary biliary cirrhosis (PBC). The objective of this study was to determine the diagnostic value of anti-KLHL12/HK1 autoantibodies in PBC. Initial discovery used sera from 22 patients with PBC and 62 non-PBC controls. KLHL12 and HK1 proteins were then analysed for immunoglobulin reactivity by immunoblot and enzyme-linked immunosorbent assay (ELISA) in two independent cohorts of PBC and disease/healthy control patients. METHODS: Serum samples from 100 patients with PBC and 165 non-PBC disease controls were analysed by immunoblot and samples from 366 patients with PBC, 174 disease controls, and 80 healthy donors were tested by ELISA. RESULTS: Anti-KLHL12 and anti-HK1 antibodies were each detected more frequently in PBC compared with non-PBC disease controls (P < 0.001). Not only are both markers highly specific for PBC (≥95%) but they also yielded higher sensitivity than anti-gp210 and anti-sp100 antibodies. Combining anti-HK1 and anti-KLHL12 with available markers (MIT3, gp210 and sp100), increased the diagnostic sensitivity for PBC. Most importantly, anti-KLHL12 and anti-HK1 antibodies were present in 10-35% of anti-mitochondrial antibody (AMA)-negative PBC patients and adding these two biomarkers to conventional PBC assays dramatically improved the serological sensitivity in AMA-negative PBC from 55% to 75% in immunoblot and 48.3% to 68.5% in ELISA. CONCLUSIONS: The addition of tests for highly specific anti-KLHL12 and anti-HK1 antibodies to AMA and ANA serological assays significantly improves efficacy in the clinical detection and diagnosis of PBC, especially for AMA-negative subjects.
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Autoanticorpos , Biomarcadores/sangue , Hexoquinase/imunologia , Cirrose Hepática Biliar/imunologia , Proteínas dos Microfilamentos/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Autoanticorpos/sangue , Autoanticorpos/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Cirrose Hepática Biliar/sangue , Análise Serial de Proteínas , Sensibilidade e EspecificidadeRESUMO
Channelrhodopsins (ChRs), which form a distinct branch of the microbial rhodopsin family, control phototaxis in green algae. Because ChRs can be expressed and function in neuronal membranes as light-gated cation channels, they have rapidly become an important optogenetic tool in neurobiology. While channelrhodopsin-2 from the unicellular alga Chlamydomonas reinhardtii (CrChR2) is the most commonly used and extensively studied optogenetic ChR, little is known about the properties of the diverse group of other ChRs. In this study, near-infrared confocal resonance Raman spectroscopy along with hydrogen-deuterium exchange and site-directed mutagenesis were used to study the structure of red-shifted ChR1 from Chlamydomonas augustae (CaChR1). These measurements reveal that (i) CaChR1 has an all-trans-retinal structure similar to those of the light-driven proton pump bacteriorhodopsin (BR) and sensory rhodopsin II but different from that of the mixed retinal composition of CrChR2, (ii) lowering the pH from 7 to 2 or substituting neutral residues for Glu169 or Asp299 does not significantly shift the ethylenic stretch frequency more than 1-2 cm(-1) in contrast to BR in which a downshift of 7-9 cm(-1) occurs reflecting neutralization of the Asp85 counterion, and (iii) the CaChR1 protonated Schiff base (SB) has stronger hydrogen bonding than BR. A model is proposed to explain these results whereby at pH 7 the predominant counterion to the SB is Asp299 (the homologue to Asp212 in BR) while Glu169 (the homologue to Asp85 in BR) exists in a neutral state. We observe an unusual constancy of the resonance Raman spectra over the broad range from pH 9 to 2 and discuss its implications. These results are in accord with recent visible absorption and current measurements of CaChR1 [Sineshchekov, O. A., et al. (2013) Intramolecular proton transfer in channelrhodopsins. Biophys. J. 104, 807-817; Li, H., et al. (2014) Role of a helix B lysine residue in the photoactive site in channelrhodopsins. Biophys. J. 106, 1607-1617].
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Chlamydomonas/química , Rodopsina/química , Rodopsinas Microbianas/química , Bases de Schiff/química , Ânions/farmacologia , Ácido Aspártico/química , Ácido Glutâmico/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Rodopsinas Sensoriais/química , Análise Espectral RamanRESUMO
RATIONALE: Rapidly performing global proteomic screens is an important goal in the post-genomic era. Correlated matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and fluorescent imaging of photocleavable peptide-coded random bead-arrays was evaluated as a critical step in a new method for proteomic screening that combines many of the advantages of MS with fluorescence-based microarrays. METHODS: Small peptide-coded model bead libraries containing up to 20 different bead species were constructed by attaching peptides to 30-34 µm diameter glass, agarose or TentaGel® beads using photocleavable biotin or a custom-designed photocleavable linker. The peptide-coded bead libraries were randomly arrayed into custom gold-coated micro-well plates with 45 µm diameter wells and subjected to fluorescence and MALDI mass spectrometric imaging (MALDI-MSI). RESULTS: Photocleavable mass-tags from individual beads in these libraries were spatially localized as ~65 µm spots using MALDI-MSI with high sensitivity and mass resolution. Fluorescently tagged beads were identified and correlated with their matching photocleavable mass-tags by comparing the fluorescence and MALDI-MS images of the same bead-array. Post-translational modification of the peptide Kemptide was also detected on individual beads in a photocleavable peptide-coded bead-array by MALDI-MSI alone, after exposure of the beads to protein kinase A (PKA). CONCLUSIONS: Correlated MALDI-MS and fluorescent imaging of photocleavable peptide-coded random bead-arrays can provide a basis for performing global proteomic screening.
Assuntos
Microesferas , Peptídeos/química , Fotólise , Espectrometria de Fluorescência/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Análise em Microsséries/instrumentação , Análise em Microsséries/métodos , Modelos Moleculares , Biblioteca de Peptídeos , Peptídeos/metabolismo , Fosforilação , Sensibilidade e Especificidade , EstreptavidinaRESUMO
Glioblastoma is a highly heterogeneous and infiltrative form of brain cancer associated with a poor outcome and limited therapeutic effectiveness. The extent of the surgery is related to survival. Reaching an accurate diagnosis and prognosis assessment by the time of the initial surgery is therefore paramount in the management of glioblastoma. To this end, we are studying the performance of SpiderMass, an ambient ionization mass spectrometry technology that can be used in vivo without invasiveness, coupled to our recently established artificial intelligence pipeline. We demonstrate that we can both stratify isocitrate dehydrogenase (IDH)-wild-type glioblastoma patients into molecular sub-groups and achieve an accurate diagnosis with over 90% accuracy after cross-validation. Interestingly, the developed method offers the same accuracy for prognosis. In addition, we are testing the potential of an immunoscoring strategy based on SpiderMass fingerprints, showing the association between prognosis and immune cell infiltration, to predict patient outcome.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Inteligência Artificial , Microambiente Tumoral , Neoplasias Encefálicas/diagnóstico , PrognósticoRESUMO
H.G. Khorana's seminal contributions to molecular biology are well-known. He also had a lesser known but still major influence on current application of advanced vibrational spectroscopic techniques such as FTIR difference spectroscopy to explore the mechanism of bacteriorhodopsin and other integral membrane proteins. In this review, I provide a personal perspective of my collaborative research and interactions with Gobind, from 1982 to 1995 when our groups published over 25 papers together which resulted in an early picture of key features of the bacteriorhodopsin proton pump mechanism. Much of this early work served as a blueprint for subsequent advances based on combining protein bioengineering and vibrational spectroscopic techniques to study integral membrane proteins.
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Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is one of the most widely used methods for imaging the spatial distribution of unlabeled small molecules such as metabolites, lipids and drugs in tissues. Recent progress has enabled many improvements including the ability to achieve single cell spatial resolution, 3D-tissue image reconstruction, and the precise identification of different isomeric and isobaric molecules. However, MALDI-MSI of high molecular weight intact proteins in biospecimens has thus far been difficult to achieve. Conventional methods normally require in situ proteolysis and peptide mass fingerprinting, have low spatial resolution, and typically detect only the most highly abundant proteins in an untargeted manner. In addition, MSI-based multiomic and multimodal workflows are needed which can image both small molecules and intact proteins from the same tissue. Such a capability can provide a more comprehensive understanding of the vast complexity of biological systems at the organ, tissue, and cellular levels of both normal and pathological function. A recently introduced top-down spatial imaging approach known as MALDI HiPLEX-IHC (MALDI-IHC for short) provides a basis for achieving this high-information content imaging of tissues and even individual cells. Based on novel photocleavable mass-tags conjugated to antibody probes, high-plex, multimodal and multiomic MALDI-based workflows have been developed to image both small molecules and intact proteins on the same tissue sample. Dual-labeled antibody probes enable multimodal mass spectrometry and fluorescent imaging of targeted intact proteins. A similar approach using the same photocleavable mass-tags can be applied to lectin and other probes. We detail here several examples of MALDI-IHC workflows designed to enable high-plex, multiomic and multimodal imaging of tissues at a spatial resolution as low as 5 µm. This approach is compared to other existing high-plex methods such as imaging mass cytometry, MIBI-TOF, GeoMx and CODEX. Finally, future applications of MALDI-IHC are discussed.
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Immunohistochemistry (IHC) combined with fluorescence microscopy provides an important and widely used tool for researchers and pathologists to image multiple biomarkers in tissue specimens. However, multiplex IHC using standard fluorescence microscopy is generally limited to 3-5 different biomarkers, with hyperspectral or multispectral methods limited to 8. We report the development of a new technology based on novel photocleavable mass-tags (PC-MTs) for facile antibody labeling, which enables highly multiplexed IHC based on MALDI mass spectrometric imaging (MALDI-IHC). This approach significantly exceeds the multiplexity of both fluorescence- and previous cleavable mass-tag-based methods. Up to 12-plex MALDI-IHC was demonstrated on mouse brain, human tonsil, and breast cancer tissues specimens, reflecting the known molecular composition, anatomy, and pathology of the targeted biomarkers. Novel dual-labeled fluorescent PC-MT antibodies and label-free small-molecule mass spectrometric imaging greatly extend the capability of this new approach. MALDI-IHC shows promise for use in the fields of tissue pathology, tissue diagnostics, therapeutics, and precision medicine.
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Biomarcadores/análise , Imuno-Histoquímica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Biomarcadores Tumorais/análise , Química Encefálica , Neoplasias da Mama/química , Feminino , Imunofluorescência , Humanos , Hibridização In Situ , Camundongos , Microesferas , Tonsila Palatina/química , Peptídeos/química , Peptídeos/efeitos da radiação , Fotoquímica , Estreptavidina , Raios UltravioletaRESUMO
Opsin-based transmembrane voltage sensors (OTVSs) are membrane proteins increasingly used in optogenetic applications to measure voltage changes across cellular membranes. In order to better understand the photophysical properties of OTVSs, we used a combination of UV-Vis absorption, fluorescence and FT-Raman spectroscopy to characterize QuasAr2 and NovArch, two closely related mutants derived from the proton pump archaerhodopsin-3 (AR3). We find both QuasAr2 and NovArch can be optically cycled repeatedly between O-like and M-like states using 5-min exposure to red (660 nm) and near-UV (405 nm) light. Longer red-light exposure resulted in the formation of a long-lived photoproduct similar to pink membrane, previously found to be a photoproduct of the BR O intermediate with a 9-cis retinylidene chromophore configuration. However, unlike QuasAr2 whose O-like state is stable in the dark, NovArch exhibits an O-like state which slowly partially decays in the dark to a stable M-like form with a deprotonated Schiff base and a 13-cis,15-anti retinylidene chromophore configuration. These results reveal a previously unknown complexity in the photochemistry of OTVSs including the ability to optically switch between different long-lived states. The possible molecular basis of these newly discovered properties along with potential optogenetic and biotechnological applications are discussed.
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Bacteriorodopsinas , Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Opsinas/metabolismo , Fotoquímica , Bombas de Próton , Análise Espectral RamanRESUMO
The crystal structure of the light-gated anion channel GtACR1 reported in our previous Research Article (Li et al., 2019) revealed a continuous tunnel traversing the protein from extracellular to intracellular pores. We proposed the tunnel as the conductance channel closed by three constrictions: C1 in the extracellular half, mid-membrane C2 containing the photoactive site, and C3 on the cytoplasmic side. Reported here, the crystal structure of bromide-bound GtACR1 reveals structural changes that relax the C1 and C3 constrictions, including a novel salt-bridge switch mechanism involving C1 and the photoactive site. These findings indicate that substrate binding induces a transition from an inactivated state to a pre-activated state in the dark that facilitates channel opening by reducing free energy in the tunnel constrictions. The results provide direct evidence that the tunnel is the closed form of the channel of GtACR1 and shed light on the light-gated channel activation mechanism.
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Channelrhodopsins/química , Ativação do Canal Iônico/fisiologia , Animais , Ânions/química , Brometos/química , Membrana Celular , Channelrhodopsins/genética , Criptófitas/química , Cristalografia por Raios X , Células HEK293 , Humanos , Transporte de Íons , Optogenética , Células Sf9RESUMO
INTRODUCTION: Breast cancer is the most diagnosed and second leading cause of cancer deaths in the U.S. female population. An estimated 5 to 10 percent of all breast cancers are inherited, caused by mutations in the breast cancer susceptibility genes (BRCA1/2). As many as 90% of all mutations are nonsense mutations, causing a truncated polypeptide product. A popular and low cost method of mutation detection has been the protein truncation test (PTT), where target regions of BRCA1/2 are PCR amplified, transcribed/translated in a cell-free protein synthesis system and analyzed for truncated polypeptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. We previously reported a novel High Throughput Solid-Phase PTT (HTS-PTT) based on an enzyme-linked immunosorbent assay (ELISA) format that eliminates the need for radioactivity, SDS-PAGE and subjective interpretation of the results. Here, we report the next generation HTS-PTT using triple-epitope-tagged proteins and demonstrate, for the first time, its efficacy on clinical genomic DNA samples for BRCA1/2 analysis. METHODS: Segments of exons 11 of BRCA1/2 open reading frames were PCR amplified from either blood derived genomic DNA or cell line mRNA. PCR primers incorporate elements for cell-free transcription/translation and epitope tagging. Cell-free expressed nascent proteins are then antibody-captured onto the wells of a microtiter plate and the relative amount of truncated polypeptide measured using antibodies against the N- and C-terminal epitope tags in an ELISA format. RESULTS: 100% diagnostic sensitivity and 96% specificity for truncating mutations in exons 11 of BRCA1/2 was achieved on one hundred blood-derived clinical genomic DNA samples which were previously assayed using the conventional gel based PTT. Feasibility of full gene coverage for BRCA1/2 using mRNA source material is also demonstrated. CONCLUSIONS: Overall, the HTS-PTT provides a simple, quantitative, objective, low cost and high throughput method for analysis of truncating mutations as an alternative to gel based PTT for BRCA analysis. The technology is readily accessible to virtually any laboratory, with the only major instrumentation required being a PCR thermocycler and a basic micro-well plate reader. When compared to conventional gel based PTT, the HTS-PTT provides excellent concordance.
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Proteína BRCA1/análise , Proteína BRCA2/análise , Neoplasias da Mama/genética , Ensaios de Triagem em Larga Escala/métodos , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Genes BRCA1 , Genes BRCA2 , Predisposição Genética para Doença , Humanos , Mutação , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genéticaRESUMO
Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Delta1-49 apolipoprotein A-I fragment (Delta49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR --> M transition. Importantly the functional bR was solubilized in discoidal bR.NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Delta49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.
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Apolipoproteína A-I/química , Proteínas de Membrana/química , Apolipoproteína A-I/genética , Bacteriorodopsinas/química , Bacteriorodopsinas/genética , Sequência de Bases , Primers do DNA/genética , Halobacterium salinarum/genética , Proteínas de Membrana/genética , Microscopia de Força Atômica , Nanopartículas/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Proteômica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Opsin-based transmembrane voltage sensors (OTVSs) are increasingly important tools for neuroscience enabling neural function in complex brain circuits to be explored in live, behaving animals. However, the visible wavelengths required for fluorescence excitation of the current generation of OTVSs limit optogenetic imaging in the brain to depths of only a few mm due to the strong absorption and scattering of visible light by biological tissues. We report that substitution of the native A1 retinal chromophore of the widely used QuasAr1/2 OTVSs with the retinal analog MMAR containing a methylamino-modified dimethylphenyl ring results in over a 100-nm redshift of the maxima of the absorption and fluorescence emission bands to near 700 and 840 nm, respectively. FT-Raman spectroscopy reveals that at pH 7 QuasAr1 with both the A1 and MMAR chromophores possess predominantly an all-trans protonated Schiff base configuration with the MMAR chromophore exhibiting increased torsion of the polyene single-/double-bond system similar to the O-intermediate of the BR photocycle. In contrast, the A1 and the MMAR chromophores of QuasAr2 exist partially in a 13-cis PSB configuration. These results demonstrate that QuasArs containing the MMAR chromophore are attractive candidates for use as NIR-OTVSs, especially for applications such as deep brain imaging.
Assuntos
Proteínas de Membrana/química , Retinaldeído/química , Análise Espectral/métodos , Sequência de Aminoácidos , OptogenéticaRESUMO
We detect internal water molecules in a membrane-embedded receptor-transducer complex and demonstrate water structure changes during formation of the signaling state. Time-resolved FTIR spectroscopy reveals stimulus-induced repositioning of one or more structurally active water molecules to a significantly more hydrophobic environment in the signaling state of the sensory rhodopsin II (SRII)-transducer (HtrII) complex. These waters, distinct from bound water molecules within the SRII receptor, appear to be in the middle of the transmembrane interface region near the Tyr199(SRII)-Asn74(HtrII) hydrogen bond. We conclude that water potentially plays an important role in the SRII --> HtrII signal transfer mechanism in the membrane's hydrophobic core.
Assuntos
Proteínas Arqueais/química , Membrana Celular/química , Halorrodopsinas/química , Domínios e Motivos de Interação entre Proteínas/fisiologia , Rodopsinas Sensoriais/química , Transdução de Sinais/fisiologia , Água/química , Proteínas Arqueais/metabolismo , Membrana Celular/metabolismo , Halorrodopsinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ligação Proteica/fisiologia , Rodopsinas Sensoriais/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Água/metabolismoRESUMO
Archaerhodopsin-3 (AR3) is a member of the microbial rhodopsin family of hepta-helical transmembrane proteins, containing a covalently bound molecule of all-trans retinal as a chromophore. It displays an absorbance band in the visible region of the solar spectrum (λmax 556 nm) and functions as a light-driven proton pump in the archaeon Halorubrum sodomense. AR3 and its mutants are widely used in neuroscience as optogenetic neural silencers and in particular as fluorescent indicators of transmembrane potential. In this study, we investigated the effect of analogs of the native ligand all-trans retinal A1 on the spectral properties and proton-pumping activity of AR3 and its single mutant AR3 (F229S). While, surprisingly, the 3-methoxyretinal A2 analog did not redshift the absorbance maximum of AR3, the analogs retinal A2 and 3-methylamino-16-nor-1,2,3,4-didehydroretinal (MMAR) did generate active redshifted AR3 pigments. The MMAR analog pigments could even be activated by near-infrared light. Furthermore, the MMAR pigments showed strongly enhanced fluorescence with an emission band in the near-infrared peaking around 815 nm. We anticipate that the AR3 pigments generated in this study have widespread potential for near-infrared exploitation as fluorescent voltage-gated sensors in optogenetics and artificial leafs and as proton pumps in bioenergy-based applications.