Alternative proteins are functional regulators in cell reprogramming by PKA activation.

It has been recently shown that many proteins are lacking from reference databases used in mass spectrometry analysis, due to their translation templated on alternative open reading frames. This questions our current understanding of gene annotation and drastically expands the theoretical proteome complexity. The functions of these alternative proteins (AltProts) still remain largely unknown. We have developed a large-scale and unsupervised approach based on cross-linking mass spectrometry (XL-MS) followed by shotgun proteomics to gather information on the functional role of AltProts by mapping them back into known signalling pathways through the identification of their reference protein (RefProt) interactors. We have identified and profiled AltProts in a cancer cell reprogramming system: NCH82 human glioma cells after 0, 16, 24 and 48 h Forskolin stimulation. Forskolin is a protein kinase A activator inducing cell differentiation and epithelial-mesenchymal transition. Our data show that AltMAP2, AltTRNAU1AP and AltEPHA5 interactions with tropomyosin 4 are downregulated under Forskolin treatment. In a wider perspective, Gene Ontology and pathway enrichment analysis (STRING) revealed that RefProts associated with AltProts are enriched in cellular mobility and transfer RNA regulation. This study strongly suggests novel roles of AltProts in multiple essential cellular functions and supports the importance of considering them in future biological studies.

Toward High Spatially Resolved Proteomics Using Expansion Microscopy.

Expansion microscopy (EM) is an emerging approach for morphological examination of biological specimens at nanoscale resolution using conventional optical microscopy. To achieve physical separation of cell structures, tissues are embedded in a swellable polymer and expanded several fold in an isotropic manner. This work shows the development and optimization of physical tissue expansion as a new method for spatially resolved large-scale proteomics. Herein we established a novel method to enlarge the tissue section to be compatible with manual microdissection on regions of interest and MS-based proteomic analysis. A major issue in expansion microscopy is the loss of protein information during the mechanical homogenization phase due to the use of proteinase K. For isotropic expansion, different homogenization agents were investigated, both to maximize protein identification and to minimize protein diffusion. Best results were obtained with SDS for homogenization. Using our modified protocol, we were able to enlarge a tissue section more than 3-fold and identified up to 655 proteins from 1 mm in size after expansion, equivalent to 330 µm in their real size corresponding thus to an average of 260 cells. This approach can be performed easily without any expensive sampling instrument. We demonstrated the compatibility of sample preparation for expansion microscopy and proteomic study in a spatial context.

The multiverse nature of epithelial to mesenchymal transition.

The epithelial mesenchymal transition (EMT) program is defined as a cellular transition from an epithelial to a mesenchymal state. This process occurs to provide the cell with new phenotypic assets and new skills to perform complex processes. EMT is regulated at multilayer levels, including transcriptional control of gene expression, regulation of RNA splicing, and translational/post-translational control. Although transcriptional regulation by EMT-inducing transcription factors (EMT-TFs), including Zeb, Snail and Slug members, is generally considered the master step in this process, emerging data indicate that all these regulatory networks may have a role in the control of EMT. There is a sort of parallelism between the biological and still unrevealed EMT complexity and the cosmological hypothesis that sustains the universe may exist as a multiverse. The presence of different EMT transition states together with the occurrence of multiple layers of regulation support the idea that EMT is just one on many out there. Is the activation of a single layer of regulation sufficient to initiate the whole EMT program? Can we postulate the activation of different EMT "dimensions"? If we think about these layers as multiple separate "universes", various scenarios can be revealed.

Optimized Sample Preparation Workflow for Improved Identification of Ghost Proteins.

Large scale proteomic strategies rely on database interrogation. Thus, only referenced proteins can be identified. Recently, Alternative Proteins (AltProts) translated from nonannotated Alternative Open reading frame (AltORFs) were discovered using customized databases. Because of their small size which confers them peptide-like physicochemical properties, they are more difficult to detect using standard proteomics strategies. In this study, we tested different preparation workflows for improving the identification of AltProts in NCH82 human glioma cell line. The highest number of identified AltProts was achieved with RIPA buffer or boiling water extraction followed by acetic acid precipitation.

Spatially-Resolved Top-down Proteomics Bridged to MALDI MS Imaging Reveals the Molecular Physiome of Brain Regions.

Tissue spatially-resolved proteomics was performed on 3 brain regions, leading to the characterization of 123 reference proteins. Moreover, 8 alternative proteins from alternative open reading frames (AltORF) were identified. Some proteins display specific post-translational modification profiles or truncation linked to the brain regions and their functions. Systems biology analysis performed on the proteome identified in each region allowed to associate sub-networks with the functional physiology of each brain region. Back correlation of the proteins identified by spatially-resolved proteomics at a given tissue localization with the MALDI MS imaging data, was then performed. As an example, mapping of the distribution of the matrix metallopeptidase 3-cleaved C-terminal fragment of α-synuclein (aa 95-140) identified its specific distribution along the hippocampal dentate gyrus. Taken together, we established the molecular physiome of 3 rat brain regions through reference and hidden proteome characterization.

Carbonic Anhydrase XII Expression Is Modulated during Epithelial Mesenchymal Transition and Regulated through Protein Kinase C Signaling.

Members of the carbonic anhydrase family are functionally involved in the regulation of intracellular and extracellular pH in physiological and pathological conditions. Their expression is finely regulated to maintain a strict control on cellular homeostasis, and it is dependent on the activation of extracellular and intracellular signaling pathways. Combining RNA sequencing (RNA-seq), NanoString, and bioinformatics data, we demonstrated that the expression of carbonic anhydrase 12 (CAXII) is significantly different in luminal and triple negative breast cancer (BC) models and patients, and is associated with the activation of an epithelial mesenchymal transition (EMT) program. In BC models, the phorbol ester 12-myristate 13-acetate (PMA)-mediated activation of protein kinase C (PKC) induced a down-regulation of CAXII with a concomitant modulation of other members of the transport metabolon, including CAIX and the sodium bicarbonate cotransporter 3 (NBCn1). This is associated with a remodeling of tumor glycolytic metabolism induced after PKC activation. Overall, this analysis highlights the dynamic nature of transport metabolom and identifies signaling pathways finely regulating this plasticity.

Proteomic expression profile of injured rat peripheral nerves revealed biological networks and processes associated with nerve regeneration.

Peripheral nerve regeneration is regulated through the coordinated spatio-temporal activation of multiple cellular pathways. In this work, an integrated proteomics and bioinformatics approach was employed to identify differentially expressed proteins at the injury-site of rat sciatic nerve at 20 days after damage. By a label-free liquid chromatography mass-spectrometry (LC-MS/MS) approach, we identified 201 differentially proteins that were assigned to specific canonical and disease and function pathways. These include proteins involved in cytoskeleton signaling and remodeling, acute phase response, and cellular metabolism. Metabolic proteins were significantly modulated after nerve injury to support a specific metabolic demand. In particular, we identified a group of proteins involved in lipid uptake and lipid storage metabolism. Immunofluorescent staining for acyl-CoA diacylglycerol acyltransferase 1 (DGAT1) and DAGT2 expression provided evidence for the expression and localization of these two isoforms in Schwann cells at the injury site in the sciatic nerve. This further supports a specific local regulation of lipid metabolism in peripheral nerve after damage.

On-tissue Direct Monitoring of Global Hydrogen/Deuterium Exchange by MALDI Mass Spectrometry: Tissue Deuterium Exchange Mass Spectrometry (TDXMS).

Hydrogen/deuterium exchange mass spectrometric (H/DXMS) methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in deuterium uptake behavior was observed when the same proteins were monitored in solution and on tissue. The higher maximum deuterium uptake at equilibrium for all proteins analyzed in solution suggests a more open conformation in the absence of interacting partners normally observed on tissue. We also demonstrate a difference in the deuterium uptake behavior of a few proteins across different morphological regions of the same tissue section. Modifications of the total number of hydrogens exchanged, as well as the kinetics of exchange, were both observed. These results provide information on the implication of protein interactions with partners as well as on the conformational changes related to these interactions, and illustrate the importance of examining protein deuterium exchange behavior in the presence of its specific microenvironment directly at the level of tissues.

Proteomic Analysis of the Spatio-temporal Based Molecular Kinetics of Acute Spinal Cord Injury Identifies a Time- and Segment-specific Window for Effective Tissue Repair.

Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target.

NanoLC-MS coupling of liquid microjunction microextraction for on-tissue proteomic analysis.

Mass spectrometry (MS)-based microproteomics on localized regions of tissue sections was achieved by direct coupling of liquid microjunction microextraction with a nanoscale liquid chromatography-tandem MS, resulting in the identification of >500 protein groups from a region as small as 250µm in diameter representing only a few hundred of cells. The method was applied on the examination of benign and tumor regions initially defined by imaging mass spectrometry (IMS) analysis of a consecutive high grade serous ovarian tumor tissue section. Results identified the higher abundance of eukaryotic translation initiation factors eIF4A, its isoform eIF4A2, and eIF5A and its isoform eIF5A2, and lower abundance of actin-binding proteins OBSCN, TAGLN and CNN3 on tumor regions, concomitant with previous findings. This demonstrates the use of the method for downstream characterization of distinct regions identified by IMS. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Integrated mass spectrometry imaging and omics workflows on the same tissue section using grid-aided, parafilm-assisted microdissection.

BACKGROUND: In spite of the number of applications describing the use of MALDI MSI, one of its major drawbacks is the limited capability of identifying multiple compound classes directly on the same tissue section. METHODS: We demonstrate the use of grid-aided, parafilm-assisted microdissection to perform MALDI MS imaging and shotgun proteomics and metabolomics in a combined workflow and using only a single tissue section. The grid is generated by microspotting acid dye 25 using a piezoelectric microspotter, and this grid was used as a guide to locate regions of interest and as an aid during manual microdissection. Subjecting the dissected pieces to the modified Folch method allows to separate the metabolites from proteins. The proteins can then be subjected to digestion under controlled conditions to improve protein identification yields. RESULTS: The proof of concept experiment on rat brain generated 162 and 140 metabolite assignments from three ROIs (cerebellum, hippocampus and midbrain/hypothalamus) in positive and negative modes, respectively, and 890, 1303 and 1059 unique proteins. Integrated metabolite and protein overrepresentation analysis identified pathways associated with the biological functions of each ROI, most of which were not identified when looking at the protein and metabolite lists individually. CONCLUSIONS: This combined MALDI MS imaging and multi-omics approach further extends the amount of information that can be generated from single tissue sections. GENERAL SIGNIFICANCE: To the best of our knowledge, this is the first report combining both imaging and multi-omics analyses in the same workflow and on the same tissue section.

Spatially-resolved protein surface microsampling from tissue sections using liquid extraction surface analysis.

Tissue microenvironment characterization presents a challenge for a better understanding of the full complexity of a pathology. Unfortunately, making a precise "picture" of the disease needs an efficient microsampling method coupled to an accurate localization for performing region-dependent proteomics. Here, we present a method that enables rapid and reproducible extraction of proteins from a tissue section to analyze a specific region at a millimeter scale. The method used a liquid-microjunction extraction with conventional detergent solution for proteomics analysis. We successfully performed immunoblotting experiments and showed the possibility to retrieve and identify more than 1400 proteins from a 1-mm diameter spot size on tissue sections with a high degree of reproducibility both qualitatively and quantitatively. Moreover, the small size of the extracted region achieved by this sampling method allows the possibility to perform multiple extractions on different tissue section points. Ten points on a sagittal rat brain tissue section were analyzed and the measured proteins clearly distinguished the different parts of the brain, thus permitting precise functional mapping. We thus demonstrate that with this technology, it is possible to map the tissue microenvironment and gain an understanding of the molecular mechanisms at millimeter resolution.

Human temporal lobe epilepsy analyses by tissue proteomics.

Although there are many types of epilepsy, temporal lobe epilepsy (TLE) is probably in humans the most common and most often studied. TLE represents 40% of the total epilepsy form of the disease and is difficult to treat. Despite a wealth of descriptive data obtained from the disease history of patients, the EEG recording, imaging techniques, and histological studies, the epileptogenic process remains poorly understood. However, it is unlikely that a single factor or a single mechanism can cause many changes associated with this neuropathological phenomenon. MALDI mass spectrometry imaging (MSI) coupled to protein identification, because of its ability to study a wide range of molecules, appears to be suitable for the preparation of molecular profiles in TLE. Seven neuropeptides have been have been identified in Dental gyrus regions of the hippocampus in relation with TLE pathology. Shot-gun studies taking into account gender influence have been performed. Tissue microextraction from control (10) toward 10 TLE patients have been analyzed after trypsin digestion followed by separation on nanoLC coupled to LTQ orbitrap. From the shot-gun analyses, results confirmed the presence of specific neuropeptides precursors and receptors in TLE patients as well as proteins involved in axons regeneration including neurotrophins, ECM proteins, cell surface proteins, membrane proteins, G-proteins, cytoskeleton proteins and tumor suppressors. Among the tumor suppressors identified, the Leucine-rich glioma inactivated 1 (LGI1) protein was found. LGI1 gene recently been demonstrated being implicated in heritability of TLE. We have also demonstrate the presence a complete profile of tumor suppressors in TLE patients, 7 have been identified. Refining this analysis taken into account the gender influence in both control and in TLE reflected the presence of specific proteins between male and female and thus mechanisms in pathology development could be completely different.

On the origin of increased sensitivity and mass resolution using silicon masks in MALDI.

Since its development, MALDI has proved its performance in the analysis of intact biomolecules up to high molecular weights, regardless of their polarity. Sensitivity of MALDI instruments is a key point for breaking the limits of observing biomolecules of lower abundances. Instrumentation is one way to improve sensitivity by increasing ion transmission and using more sensitive detection systems. On the other side, improving MALDI ion production yields would have important outcomes. MALDI ion production is still not well-controlled and, indeed, the amount of ions produced per laser shot with respect to the total volume of desorbed material is very low. This has particular implications for certain applications, such as MALDI MS imaging where laser beam focusing as fine as possible (5-10 µm) is searched in order to reach higher spatial resolution images. However, various studies point out an intrinsic decrease in signal intensity for strong focusing. We have therefore been interested in developing silicon mask systems to decrease an irradiated area by cutting rather than focusing the laser beam and to study the parameters affecting sensitivity using such systems. For this, we systematically examined variation with laser fluence of intensity and spectral resolution in MALDI of standard peptides when using silicon-etched masks of various aperture sizes. These studies demonstrate a simultaneous increase in spectral resolution and signal intensity. Origin of this effect is discussed in the frame of the two-step ionization model. Experimental data in the low fluence range are fitted with an increase of the primary ionization through matrix-silicon edge contact provided by the masks. On the other hand, behavior at higher fluence could be explained by an effect on the secondary ionization via changes in the plume dynamics.

Calreticulin contributes to C1q-dependent recruitment of microglia in the leech Hirudo medicinalis following a CNS injury.

BACKGROUND: The medicinal leech is considered as a complementary and appropriate model to study immune functions in the central nervous system (CNS). In a context in which an injured leech's CNS can naturally restore normal synaptic connections, the accumulation of microglia (immune cells of the CNS that are exclusively resident in leeches) has been shown to be essential at the lesion to engage the axonal sprouting. HmC1q (Hm for Hirudo medicinalis) possesses chemotactic properties that are important in the microglial cell recruitment by recognizing at least a C1q binding protein (HmC1qBP alias gC1qR). MATERIAL AND METHODS: Recombinant forms of C1q were used in affinity purification and in vitro chemotaxis assays. Anti-calreticulin antibodies were used to neutralize C1q-mediated chemotaxis and locate the production of calreticulin in leech CNS. RESULTS: A newly characterized leech calreticulin (HmCalR) has been shown to interact with C1q and participate to the HmC1q-dependent microglia accumulation. HmCalR, which has been detected in only some microglial cells, is consequently a second binding protein for HmC1q, allowing the chemoattraction of resident microglia in the nerve repair process. CONCLUSIONS: These data give new insight into calreticulin/C1q interaction in an immune function of neuroprotection, suggesting another molecular target to use in investigation of microglia reactivity in a model of CNS injury.

Quantification-based mass spectrometry imaging of proteins by parafilm assisted microdissection.

MALDI mass spectrometry imaging (MALDI-MSI) was presented as a good strategy to highlight regions presenting specific phenotypes based on molecular content. The proteins present in the different areas can be identified by MALDI MSI; however, the number of protein identifications remains low in comparison with classical MS-based proteomics approaches. To overcome this, a new strategy, involving the microdissection of tissue sections mounted on parafilm M-covered glass slides, is presented. Extraction and fractionation of proteins from a specific region of interest were investigated, leading to the identification of more than 1000 proteins from each microdissected piece. The strength of this cheap technique lies in the facile excision of millimeter-sized portions from the tissue allowing for the identification of proteins from cells of a specific phenotype obtained from the MALDI MS imaging-based molecular classification using hierarchical clustering. This approach can be extended to whole tissue sections in order to generate images of the section based on label-free quantification obtained from identification data. As a proof of concept, we have studied a tissue mounted on a parafilm M-covered glass slide, cut it into regular pieces, and submitted each piece to identification and quantification according to the developed parafilm-assisted microdissection (PAM) method. Images were then reconstructed by relative quantification of identified proteins based on spectral counting of the peptides analyzed by nanoLC-MS and MS/MS. This strategy of quantification-based MSI offers new possibilities for mapping a large number of high and low abundance proteins.

Ionic matrices pre-spotted matrix-assisted laser desorption/ionization plates for patient maker following in course of treatment, drug titration, and MALDI mass spectrometry imaging.

In the current study, we compared plastic matrix-assisted laser desorption/ionization (MALDI) plates pre-spotted with different solid ionic matrices. Data reflect that after 3 months of storage, the standards were oxidized in α-cyano-4-hydroxycinnamic acid (HCCA) whether or not in HCCA/3-acetylpyridine (3APY) and HCCA/aniline, and certain peptides, such as ubiquitin, were not detected using the HCCA matrix, whereas they were detected in pre-spotted ionic matrices. Application in peptidomics of these MALDI matrices pre-spotted plates (after 3 months of storage) with ovarian cyst fluid showed less intense signals with HCCA than with solid ionic matrices. We show that these pre-spotted ionic matrices plates can be used for relative drug quantification, high mass protein detection, and MALDI mass spectrometry imaging.

Systematic compositional analysis of exosomal extracellular vesicles produced by cells undergoing apoptosis, necroptosis and ferroptosis.

Formation of extracellular vesicles (EVs) has emerged as a novel paradigm in cell-to-cell communication in health and disease. EVs are notably produced during cell death but it had remained unclear whether different modalities of regulated cell death (RCD) influence the biogenesis and composition of EVs. To this end, we performed a comparative analysis of steady-state (ssEVs) and cell death-associated EVs (cdEVs) following TNF-induced necroptosis (necEVs), anti-Fas-induced apoptosis (apoEVs), and ML162-induced ferroptosis (ferEVs) using the same cell line. For each RCD condition, we determined the biophysical and biochemical characteristics of the cell death-associated EVs (cdEVs), the protein cargo, and the presence of methylated ribosomal RNA. We found that the global protein content of all cdEVs was increased compared to steady-state EVs. Qualitatively, the isolated exosomal ssEVs and cdEVs, contained a largely overlapping protein cargo including some quantitative differences in particular proteins. All cdEVs were enriched for proteins involved in RNA splicing and nuclear export, and showed distinctive rRNA methylation patterns compared to ssEVs. Interestingly, necEVs and apoEVs, but strikingly not ferEVs, showed enrichment of proteins involved in ribosome biogenesis. Altogether, our work documents quantitative and qualitative differences between ssEVs and cdEVs.

Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer.

The large amount of data generated using matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) poses a challenge for data analysis. In fact, generally about 1.10(8)-1.10(9) values (m/z, I) are stored after a single MALDI-MSI experiment. This imposes processing techniques using dedicated informatics tools to be used since manual data interpretation is excluded. This work proposes and summarizes an approach that utilizes a multivariable analysis of MSI data. The multivariate analysis, such as principal component analysis-symbolic discriminant analysis, can remove and highlight specific m/z from the spectra in a specific region of interest. This approach facilitates data processing and provides better reproducibility, and thus, broadband acquisition for MALDI-MSI should be considered an effective tool to highlight biomarkers of interest. Additionally, we demonstrate the importance of the hierarchical classification of biomarkers by analyzing studies of clusters obtained either from digested or undigested tissues and using bottom-up and in-source decay strategies for in-tissue protein identification. This provides the possibility for the rapid identification of specific markers from different histological samples and their direct localization in tissues. We present an example from a prostate cancer study using formalin-fixed paraffin-embedded tissue.

MALDI imaging mass spectrometry: state of the art technology in clinical proteomics.

A decade after its inception, MALDI imaging mass spectrometry has become a unique technique in the proteomics arsenal for biomarker hunting in a variety of diseases. At this stage of development, it is important to ask whether we can consider this technique to be sufficiently developed for routine use in a clinical setting or an indispensable technology used in translational research. In this report, we consider the contributions of MALDI imaging mass spectrometry and profiling technologies to clinical studies. In addition, we outline new directions that are required to align these technologies with the objectives of clinical proteomics, including: 1) diagnosis based on profile signatures that complement histopathology, 2) early detection of disease, 3) selection of therapeutic combinations based on the individual patient's entire disease-specific protein network, 4) real time assessment of therapeutic efficacy and toxicity, 5) rational redirection of therapy based on changes in the diseased protein network that are associated with drug resistance, and 6) combinatorial therapy in which the signaling pathway itself is viewed as the target rather than any single "node" in the pathway.