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.
The recent completion of the swine genome sequencing project and development of a high density porcine SNP array has made genome-wide association (GWA) studies feasible in pigs.
Using Illumina's ...PorcineSNP60 BeadChip, we performed a pilot GWA study in 820 commercial female pigs phenotyped for backfat, loin muscle area, body conformation in addition to feet and leg (FL) structural soundness traits. A total of 51,385 SNPs were jointly fitted using Bayesian techniques as random effects in a mixture model that assumed a known large proportion (99.5%) of SNPs had zero effect. SNP annotations were implemented through the Sus scrofa Build 9 available from pig Ensembl. We discovered a number of candidate chromosomal regions, and some of them corresponded to QTL regions previously reported. We not only have identified some well-known candidate genes for the traits of interest, such as MC4R (for backfat) and IGF2 (for loin muscle area), but also obtained novel promising genes, including CHCHD3 (for backfat), BMP2 (for loin muscle area, body size and several FL structure traits), and some HOXA family genes (for overall leg action). The candidate regions responsible for body conformation and FL structure soundness did not overlap greatly which implied that these traits were controlled by different genes. Functional clustering analyses classified the genes into categories related to bone and cartilage development, muscle growth and development or the insulin pathway suggesting the traits are regulated by common pathways or gene networks that exert roles at different spatial and temporal stages.
This study is one of the earliest GWA reports on important quantitative traits in pigs, and the findings will contribute to the further biological function analysis of the identified candidate genes and potential utilization of them in marker assisted selection.
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.
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
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.
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.
Microbial rhodopsins have become an important tool in the field of optogenetics. However, effective in vivo optogenetics is in many cases severely limited due to the strong absorption and scattering ...of visible light by biological tissues. Recently, a combination of opsin site-directed mutagenesis and analog retinal substitution has produced variants of proteorhodopsin which absorb maximally in the near-infrared (NIR). In this study, UV-Visible-NIR absorption and resonance Raman spectroscopy were used to study the double mutant, D212N/F234S, of green absorbing proteorhodopsin (GPR) regenerated with MMAR, a retinal analog containing a methylamino modified β-ionone ring. Four distinct subcomponent absorption bands with peak maxima near 560, 620, 710 and 780 nm are detected with the NIR bands dominant at pH <7.3, and the visible bands dominant at pH 9.5. FT-Raman using 1064-nm excitation reveal two strong ethylenic bands at 1482 and 1498 cm-1 corresponding to the NIR subcomponent absorption bands based on an extended linear correlation between λmax and γC = C. This spectrum exhibits two intense bands in the fingerprint and HOOP mode regions that are highly characteristic of the O640 photointermediate from the light-adapted bacteriorhodopsin photocycle. In contrast, 532-nm excitation enhances the 560-nm component, which exhibits bands very similar to light-adapted bacteriorhodopsin and/or the acid-purple form of bacteriorhodopsin. Native GPR and its mutant D97N when regenerated with MMAR also exhibit similar absorption and Raman bands but with weaker contributions from the NIR absorbing components. Based on these results it is proposed that the NIR absorption in GPR-D212N/F234S with MMAR arises from an O-like chromophore, where the Schiff base counterion D97 is protonated and the MMAR adopts an all-trans configuration with a non-planar geometry due to twists in the conjugated polyene segment. This configuration is characterized by extensive charge delocalization, most likely involving nitrogens atoms in the MMAR chromophore.
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.
Graphical Abstract
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.
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.
The proton pump archaerhodopsin‐3 (AR3) is widely used in optogenetics as a silencer of neural activity or as a fluorescent indicator of transmembrane potential. In this research, we utilize analogs of its native chromophore all‐trans retinal to generate variants of AR3 and a single mutant. One analog from this study generated redshifted AR3 pigments which could be activated by near‐infrared light. Furthermore, these pigments showed strongly enhanced fluorescence with an emission band in the near‐infrared. We anticipate that these analog AR3 pigments have potential for exploiting near‐infrared light in several optogenetic and bioenergy‐based applications.
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.
Opsin‐based transmembrane voltage sensors (OTVSs) are NIR fluorescence emitting membrane proteins increasingly used in optogenetic applications to measure voltage changes across cellular membranes for instance in brain imaging studies. A combination of UV‐Vis absorption, fluorescence and FT‐Raman spectroscopy was used to characterize QuasAr2 and NovArch, two closely related OTVSs derived from the proton pump archaerhodopsin‐3. Both QuasAr2 and NovArch can be optically switched repeatedly between long‐lived O‐like and M‐like states revealing a previously unknown complexity in the photochemistry of these OTVSs.
PDF
