Individual Protein Species Research Articles

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins of organelles, cellular signaling, bioenergetic metabolism and molecular chaperoning.

During the natural aging process, frailty is often associated with abnormal muscular performance. Although inter-individual differences exit, in most elderly the tissue mass and physiological functionality of voluntary muscles drastically decreases. In order to study age-related contractile decline, animal model research is of central importance in the field of biogerontology. Here we have analyzed wild type mouse muscle to establish a proteomic map of crude tissue extracts. Proteomics is an advanced and large-scale biochemical method that attempts to identify all accessible proteins in a given biological sample. It is a technology-driven approach that uses mass spectrometry for the characterization of individual protein species. Total protein extracts were used in this study in order to minimize the potential introduction of artefacts due to excess subcellular fractionation procedures. In this report, the proteomic survey of aged muscles has focused on organellar marker proteins, as well as proteins that are involved in cellular signaling, the regulation of ion homeostasis, bioenergetic metabolism and molecular chaperoning. Hence, this study has establish a proteomic reference map of a highly suitable model system for future aging research.

Field model for multistate lateral diffusion of various transmembrane proteins observed in living Dictyostelium cells.

The lateral diffusion of transmembrane proteins on plasma membranes is a fundamental process for various cellular functions. Diffusion properties specific for individual protein species have been extensively studied, but the common features among protein species are poorly understood. Here, we systematically studied the lateral diffusion of various transmembrane proteins in the lower eukaryote Dictyostelium discoideum cells using a hidden Markov model for single-molecule trajectories obtained experimentally. As common features, all membrane proteins that had from one to ten transmembrane regions adopted three free diffusion states with similar diffusion coefficients regardless of their structural variability. All protein species reduced their mobility similarly upon the inhibition of microtubule or actin cytoskeleton dynamics, or myosin II. The relationship between protein size and the diffusion coefficient was consistent with the Saffman-Delbrück model, meaning that membrane viscosity is a major determinant of lateral diffusion, but protein size is not. These protein species-independent properties of multistate free diffusion were explained simply and quantitatively by free diffusion on the three membrane regions with different viscosities, which is in sharp contrast to the complex diffusion behavior of transmembrane proteins in higher eukaryotes.

Verification of Protein Changes Determined by 2D-DIGE Based Proteomics Using Immunofluorescence Microscopy.

Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a key biochemical method for the comparative analysis of complex protein mixtures. The technique focuses on the identification and characterization of individual protein species following gel electrophoretic separation making it an important analytical tool of top-down proteomics. In order to verify changes in the expression levels of a particular protein, as determined by 2D-DIGE analysis, and evaluate the subcellular localization of the proteoform of interest, immunofluorescence microscopy is very well suited. This chapter describes in detail the preparation of tissue specimens and the process of cryo-sectioning, as well as incubation with primary antibodies and fluorescently labeled secondary antibodies, followed by image analysis. As illustrative examples, the co-detection of immuno-labeled dystrophin and the Y-chromosome in skeletal muscle are shown, and the localization of calbindin in the cerebellum is presented.

Two-Dimensional Gel Electrophoresis and 2D-DIGE.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to be one of the most versatile and widely used techniques to study the proteome of a biological system, particularly in the separation of intact proteins. A modified version of 2D-PAGE, two-dimensional difference gel electrophoresis (2D-DIGE), which uses differential labeling of protein samples with up to three fluorescent tags, offers greater sensitivity and reproducibility over conventional 2D-PAGE gels for differential quantitative analysis of protein expression between experimental groups. Both these methods have distinct advantages in the separation and identification of thousands of individual protein species including protein isoforms and post-translational modifications. This chapter discusses the principles of 2D-PAGE and 2D-DIGE including limitations to the methods. 2D-PAGE and 2D-DIGE continue to be popular methods in bioprocessing-related research, particularly on recombinant Chinese hamster ovary cells, which are also discussed in this chapter.

Zinc-finger antiviral protein (ZAP) is a restriction factor for replication of modified vaccinia virus Ankara (MVA) in human cells.

Modified vaccinia virus Ankara (MVA) is an approved smallpox vaccine and a promising vaccine vector for other pathogens as well as for cancer therapeutics with more than 200 current or completed clinical trials. MVA was derived by passaging the parental Ankara vaccine virus hundreds of times in chick embryo fibroblasts during which it lost the ability to replicate in human and most other mammalian cells. Although this replication deficiency is an important safety feature, the genetic basis of the host restriction is not understood. Here, an unbiased human genome-wide RNAi screen in human A549 cells revealed that the zinc-finger antiviral protein (ZAP), previously shown to inhibit certain RNA viruses, is a host restriction factor for MVA, a DNA virus. Additional studies demonstrated enhanced MVA replication in several human cell lines following knockdown of ZAP. Furthermore, CRISPR-Cas9 knockout of ZAP in human A549 cells increased MVA replication and spread by more than one log but had no effect on a non-attenuated strain of vaccinia virus. The intact viral C16 protein, which had been disrupted in MVA, antagonized ZAP by binding and sequestering the protein in cytoplasmic punctate structures. Studies aimed at exploring the mechanism by which ZAP restricts MVA replication in the absence of C16 showed that knockout of ZAP had no discernible effect on viral DNA or individual mRNA or protein species as determined by droplet digital polymerase chain reaction, deep RNA sequencing and mass spectrometry, respectively. Instead, inactivation of ZAP reduced the number of aberrant, dense, spherical particles that typically form in MVA-infected human cells, suggesting that ZAP has a novel role in interfering with a late step in the assembly of infectious MVA virions in the absence of the C16 protein.

Single and Combined Methods to Specifically or Bulk-Purify RNA-Protein Complexes.

The ribonome interconnects the proteome and the transcriptome. Specific biology is situated at this interface, which can be studied in bulk using omics approaches or specifically by targeting an individual protein or RNA species. In this review, we focus on both RNA- and ribonucleoprotein-(RNP) centric methods. These methods can be used to study the dynamics of the ribonome in response to a stimulus or to identify the proteins that interact with a specific RNA species. The purpose of this review is to provide and discuss an overview of strategies to cross-link RNA to proteins and the currently available RNA- and RNP-centric approaches to study RNPs. We elaborate on some major challenges common to most methods, involving RNP yield, purity and experimental cost. We identify the origin of these difficulties and propose to combine existing approaches to overcome these challenges. The solutions provided build on the recently developed organic phase separation protocols, such as Cross-Linked RNA eXtraction (XRNAX), orthogonal organic phase separation (OOPS) and Phenol-Toluol extraction (PTex).

In Silico Evidence That Protein Unfolding is a Precursor of Protein Aggregation.

We present a computational study on the folding and aggregation of proteins in an aqueous environment, as a function of its concentration. We show how the increase of the concentration of individual protein species can induce a partial unfolding of the native conformation without the occurrence of aggregates. A further increment of the protein concentration results in the complete loss of the folded structures and induces the formation of protein aggregates. We discuss the effect of the protein interface on the water fluctuations in the protein hydration shell and their relevance in the protein-protein interaction.

FLASHDeconv: Ultrafast, High-Quality Feature Deconvolution for Top-Down Proteomics

Top-down mass spectrometry (TD-MS)-based proteomics analyzes intact proteoforms and thus preserves information about individual protein species. The MS signal of these high-mass analytes is complex and challenges the accurate determination of proteoform masses. Fast and accurate feature deconvolution (i.e., the determination of intact proteoform masses) is, therefore, an essential step for TD data analysis. Here, we present FLASHDeconv, an algorithm achieving higher deconvolution quality, with an execution speed two orders of magnitude faster than existing approaches. FLASHDeconv transforms peak positions (m/z) within spectra into log m/z space. This simple transformation turns the deconvolution problem into a search for constant patterns, thereby greatly accelerating the process. In both simple and complex samples, FLASHDeconv reports more genuine feature masses and substantially fewer artifacts than other existing methods. FLASHDeconv is freely available for download here: https://www.openms.org/flashdeconv/. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.

Multiomic approaches to delineate the pathogenesis of cardiac disease.

Comprehensive analyses of the genome, transcriptome, proteome and metabolome are instrumental in identifying biomarkers of disease, to gain insight into mechanisms underlying the development of cardiovascular disease, and show promise for better stratifying patients according to disease subtypes. This review highlights recent 'omics' studies, including integration of multiple 'omics' that have advanced mechanistic understanding and diagnosis in humans and animal models. Transcriptome-based discovery continues to be a primary method to obtain data for hypothesis generation and the understanding of disease pathogenesis has been enhanced by single cell-based methods capable of revealing heterogeneity in cellular responses. Advances in proteome coverage and quantitation of individual protein species, together with enhanced methods for detecting posttranslational modifications, have improved discovery of protein-based biomarkers. High-throughput assays capable of quantitating the vast majority of any particular type of biomolecule within a tissue sample, isolated cells or plasma are now available. In order to make best use of the large amount of data that can be generated on given molecule types, as well as their interrelationships in disease, continued development of pattern-recognition algorithms ('machine learning') will be required and the subclassification of disease that is made possible by such algorithms will be likely to inform clinical practice, and vice versa.

Magnetic Particle-Based Immunoprecipitation of Nanoscale Extracellular Vesicles from Biofluids.

Analysis of nanoscale extracellular vesicles (nsEVs) present in blood, cell culture media, and other biofluids has shown tremendous promise in enabling the development of noninvasive blood-based clinical diagnostic tests, predicting and monitoring the efficacy of treatment programs, and providing molecular level insights into pathology that can enlighten new drug targets in the contexts of health conditions such as cancer and Alzheimer's Disease (AD). In this chapter, we present methods for using magnetic particle-based immunoprecipitation to enrich highly purified populations of nsEVs directly from plasma, serum, and other biofluids. These methods enable downstream analysis of nsEV protein and nucleic acid constituents in the contexts of both global omics profiling and quantification of individual protein or nucleic acid species of interest. Additionally, these methods allow the researcher to either enrich total nsEV populations or enrich nsEVs derived from a particular tissue type from the overall nsEV population. The methods described here are compatible with parallel processing of dozens of biofluid samples and can be valuable tools for enabling nsEV analyses that have high translational relevance in the development of both novel therapeutics and noninvasive diagnostic assays.

Comparative Proteomics of Rubber Latex Revealed Multiple Protein Species of REF/SRPP Family Respond Diversely to Ethylene Stimulation among Different Rubber Tree Clones.

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are two key factors for natural rubber biosynthesis. To further understand the roles of these proteins in rubber formation, six different genes for latex abundant REF or SRPP proteins, including REF138,175,258 and SRPP117,204,243, were characterized from Hevea brasiliensis Reyan (RY) 7-33-97. Sequence analysis showed that REFs have a variable and long N-terminal, whereas SRPPs have a variable and long C-terminal beyond the REF domain, and REF258 has a β subunit of ATPase in its N-terminal. Through two-dimensional electrophoresis (2-DE), each REF/SRPP protein was separated into multiple protein spots on 2-DE gels, indicating they have multiple protein species. The abundance of REF/SRPP proteins was compared between ethylene and control treatments or among rubber tree clones with different levels of latex productivity by analyzing 2-DE gels. The total abundance of each REF/SRPP protein decreased or changed a little upon ethylene stimulation, whereas the abundance of multiple protein species of the same REF/SRPP changed diversely. Among the three rubber tree clones, the abundance of the protein species also differed significantly. Especially, two protein species of REF175 or REF258 were ethylene-responsive only in the high latex productivity clone RY 8-79 instead of in RY 7-33-97 and PR 107. Some individual protein species were positively related to ethylene stimulation and latex productivity. These results suggested that the specific protein species could be more important than others for rubber production and post-translational modifications might play important roles in rubber biosynthesis.

Co-detection of Target and Total Protein by CyDye Labeling and Fluorescent ECL Plex Immunoblotting in a Standard Proteomics Workflow.

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis have been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with ECL Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27 kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.

Geometry of membrane fission

Cellular membranes define the functional geometry of intracellular space. Formation of new membrane compartments and maintenance of complex organelles require division and disconnection of cellular membranes, a process termed membrane fission. Peripheral membrane proteins generally control membrane remodeling during fission. Local membrane stresses, reflecting molecular geometry of membrane-interacting parts of these proteins, sum up to produce the key membrane geometries of fission: the saddle-shaped neck and hour-glass hemifission intermediate. Here, we review the fundamental principles behind the translation of molecular geometry into membrane shape and topology during fission. We emphasize the central role the membrane insertion of specialized protein domains plays in orchestrating fission in vitro and in cells. We further compare individual to synergistic action of the membrane insertion during fission mediated by individual protein species, proteins complexes or membrane domains. Finally, we describe how local geometry of fission intermediates defines the functional design of the protein complexes catalyzing fission of cellular membranes.

Conditional independence mapping of DIGE data reveals PDIA3 protein species as key nodes associated with muscle aerobic capacity.

Profiling of protein species is important because gene polymorphisms, splice variations and post-translational modifications may combine and give rise to multiple protein species that have different effects on cellular function. Two-dimensional gel electrophoresis is one of the most robust methods for differential analysis of protein species, but bioinformatic interrogation is challenging because the consequences of changes in the abundance of individual protein species on cell function are unknown and cannot be predicted. We conducted DIGE of soleus muscle from male and female rats artificially selected as either high- or low-capacity runners (HCR and LCR, respectively). In total 696 protein species were resolved and LC–MS/MS identified proteins in 337 spots. Forty protein species were differentially (P < 0.05, FDR < 10%) expressed between HCR and LCR and conditional independence mapping found distinct networks within these data, which brought insight beyond that achieved by functional annotation. Protein disulphide isomerase A3 emerged as a key node segregating with differences in aerobic capacity and unsupervised bibliometric analysis highlighted further links to signal transducer and activator of transcription 3, which were confirmed by western blotting. Thus, conditional independence mapping is a useful technique for interrogating DIGE data that is capable of highlighting latent features.

The Future of Biotechnology

Rational cell engineering aims to increase the quality and quantity of the manufactured products by developing improved cell lines. Previously the selection for improvements has been carried out on a trial and error basis: cells that produced high levels of the desired product were positively selected and kept, while low–producing lines were discarded. Little or no understanding of the underlying biological and molecular processes was applied in this selection. However, with the increasing availability of new tools a more informed process is now possible. Most of these tools developed in the past decade aim to understand the biological make–up of cells by employing high–throughput (HT) approaches. For example, the HT of DNA sequencing allows the analysis of the genome of a specific cell line (genomics), RNA microarrays give information about the different types of mRNAs in a cell (transcriptomics), HT analysis of the entire protein contents shows the individual protein species present in the cell (proteomics). The information that these –omics approaches provide can be very useful for a detailed understanding of the cell factories and hence their improvement for the manufacturing of desired products.

MALDI-imaging segmentation is a powerful tool for spatial functional proteomic analysis of human larynx carcinoma

For several decades, conventional histological staining and immunohistochemistry (IHC) have been the main tools to visualize and understand tissue morphology and structure. IHC visualizes the spatial distribution of individual protein species directly in tissue. However, a specific antibody is required for each protein, and multiplexing capabilities are extremely limited, rarely visualizing more than two proteins simultaneously. With the recent emergence of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-imaging), it is becoming possible to study more complex proteomic patterns directly in tissue. However, the analysis and interpretation of large and complex MALDI-imaging data requires advanced computational methods. In this paper, we show how the recently introduced method of spatial segmentation can be applied to analysis and interpretation of a larynx carcinoma section and compare the spatial segmentation with the histological annotation of the same tissue section. Matrix-assisted laser desorption/ionization imaging is a label-free spatially resolved analytical technique, which allows detection and visualization of hundreds of proteins at once. Spatial segmentation of the MALDI-imaging data by clustering of spectra by their similarity was performed, automatically generating a spatial segmentation map of the tissue section, where regions of similar proteomic patterns were highlighted. The tissue was stained with the hematoxylin and eosin (H&E), histopathologically analyzed and annotated. The segmentation map was interpreted after its overlay with the H&E microscopy image. The automatically generated segmentation map exhibits high correspondence to the detailed histological annotation of the larynx carcinoma tissue section. By superimposing, the segmentation map based on the proteomic profiles with H&E-stained microscopic images, we demonstrate precise localization of complex and histopathologically relevant tissue features in an automated way. The combination of MALDI-imaging and automatic spatial segmentation is a useful approach in analyzing carcinoma tissue and provides a deeper insight into the functional proteomic organization of the respective tissue.

Quantitative phosphoproteomics reveals link between Helicobacter pylori infection and RNA splicing modulation in host cells

The Gram-negative, spiral-shaped bacterium Helicobacter pylori is a common human pathogen that causes chronic inflammation of the human gastric mucosa, leading to peptic ulceration and/or gastric cancer. Here, we analyzed changes in the phosphoproteome of gastric epithelial cells (AGS) upon infection with H. pylori using a combination of SILAC, phosphoprotein enrichment, 2-DE, and MALDI TOF/TOF-MS. From a total of 526 spots we identified 391 protein species (143 proteins) and quantified 332 (127 proteins). Nearly, one-third of the identified proteins (40/143) were associated with the spliceosome or RNA splicing. The abundance of 20 proteins was altered by H. pylori infection, in particular, a number of serine arginine-rich (SR) proteins involved in the regulation and control of alternative splicing. Importantly, the combined methodologies enabled the detection of infection-dependent protein species-specific regulation, suggesting functional modulation of individual protein species. These findings reveal unexpected new insights into the mechanisms of host cell manipulation by H. pylori, which are likely associated with gastric pathologies, including gastric cancer.

Conformer-Specific Hydrogen Exchange Analysis of Aβ(1–42) Oligomers by Top-Down Electron Capture Dissociation Mass Spectrometry

Protein structural studies are particularly challenging under conditions in which several conformational species (e.g., monomers and aggregated forms) coexist in solution. Most spectroscopic techniques provide population-averaged data. Hence, it is usually not possible to obtain detailed structural information on individual protein species in heterogeneous samples. The current work employs an experimental strategy that addresses this issue. Solution-phase hydrogen exchange (HX) is used in combination with tandem mass spectrometry. Electrosprayed intact ions exhibiting specific HX mass shifts are selected in the gas phase, followed by electron capture dissociation. The resulting fragment ion deuteration pattern provides amide hydrogen bonding information in a conformer-specific and spatially resolved fashion. The feasibility of this approach is demonstrated by applying it to neurotoxic Aβ(1-42) oligomers that coexist with disordered monomers in solution. The findings of this study point to similarities between oligomers and mature amyloid fibrils with regard to the Aβ(1-42) backbone organization. Specifically, fibrils and oligomers appear to share a β-loop-β secondary structure motif. The spatial resolution obtained with the "top-down" approach used here exceeds that of earlier proteolysis-based HX data on Aβ.

Finding one's way in proteomics: a protein species nomenclature

Our knowledge of proteins has greatly improved in recent years, driven by new technologies in the fields of molecular biology and proteome research. It has become clear that from a single gene not only one single gene product but many different ones - termed protein species - are generated, all of which may be associated with different functions. Nonetheless, an unambiguous nomenclature for describing individual protein species is still lacking. With the present paper we therefore propose a systematic nomenclature for the comprehensive description of protein species. The protein species nomenclature is flexible and adaptable to every level of knowledge and of experimental data in accordance with the exact chemical composition of individual protein species. As a minimum description the entry name (gene name + species according to the UniProt knowledgebase) can be used, if no analytical data about the target protein species are available.

Multi-crystallin complexes exist in the water-soluble high molecular weight protein fractions of aging normal and cataractous human lenses

The purpose of the study was to identify non-covalently held complexes that exist in the water-soluble high molecular weight (WS-HMW) protein fractions of normal human lenses of 20-year-old and 60- to 70-year-old, and in the age-matched 60- to 70-year-old cataractous lenses. The WS protein fractions were prepared from five pooled normal lenses of 20-year-old donors or five pooled lenses of 60- to 70-year-old donors or four pooled cataractous lenses (with nuclear opacity) of 60- to 70-year-old donors. Each WS protein fraction was subjected to size-exclusion chromatography using an Agarose A 5m column to recover the void volume WS-HMW protein fraction. A method known as blue-native polyacrylamide gel electrophoresis (BN-PAGE), which allows the isolation of large multi-protein complexes (MPCs) in their native state for further characterization, was used to separate such complexes from individual WS-HMW protein fractions. The protein species that existed as a complex were excised from a gel and trypsin-digested, and the amino acid sequences of the tryptic fragments analyzed by electrospray tandem mass spectrometry (ES-MS/MS). After the second-dimensional sodium dodecyl sulfate–PAGE during BN-PAGE, protein complexes containing a total of 16, 12, and 24 species with M r between 10 and 90 kDa were identified in the HMW protein fractions of normal lenses of 20-year-old, 60- to 70-year-old and cataractous lenses of 60- to 70-year-old donors, respectively. Based on the amino acid sequences of tryptic peptides of individual protein species in the complexes by the ES-MS/MS method, the presence of α-, β-, and γ-crystallin species along with beaded filament proteins (filensin and phakinin) was observed in the 20-year-old normal lenses. The 60- to 70-year-old normal lenses contained filensin and aldehyde dehydrogenase in addition to the above crystallins. Similarly, the age-matched cataractous lenses also contained the above crystallins and aldehyde dehydrogenase but lacked beaded filament proteins. Protein complexes, held mostly via non-covalent bonding, were seen in the WS-HMW proteins of 20-year-old normal, 60- to 70-year-old normal, and 60- to 70-year-old cataractous lenses. The complexes in the normal lenses were made of α-, β-, and γ-crystallin species, beaded filament proteins (filensin and/or phakinin), and aldehyde dehydrogenase. The complexes in the age-matched cataractous lenses also contained these crystallins, and aldehyde dehydrogenase, but not the beaded filament proteins. Further, the crystallin fragments were greater in number in the cataractous lenses compared to the age-matched normal lenses. During multi-angle light scattering (MALS), the HMW proteins from cataractous lenses exhibited species with lower molecular weight range than age-matched normal lenses. The HMW protein preparations from both normal and cataractous lenses showed spherical structures on electron microscopic analysis.