Histone H3 clipping is a novel signature of human neutrophil extracellular traps.

Neutrophils are critical to host defence, executing diverse strategies to perform their antimicrobial and regulatory functions. One tactic is the production of neutrophil extracellular traps (NETs). In response to certain stimuli, neutrophils decondense their lobulated nucleus and release chromatin into the extracellular space through a process called NETosis. However, NETosis, and the subsequent degradation of NETs, can become dysregulated. NETs are proposed to play a role in infectious as well as many non-infection related diseases including cancer, thrombosis, autoimmunity and neurological disease. Consequently, there is a need to develop specific tools for the study of these structures in disease contexts. In this study, we identified a NET-specific histone H3 cleavage event and harnessed this to develop a cleavage site-specific antibody for the detection of human NETs. By microscopy, this antibody distinguishes NETs from chromatin in purified and mixed cell samples. It also detects NETs in tissue sections. We propose this antibody as a new tool to detect and quantify NETs.

The comparison between 2DE-MS and bottom-up LC-MS demands high-end techniques for both technologies.

In contrast to bottom-up LC-MS only 2DE-MS can separate and detect a huge number of human protein species. Kwiatkowski et al. (in this issue) established parameters to estimate the amount of protein speciation for each human protein. Proteins identified in 2DE-MS approaches showed more protein speciation than in bottom-up LC-MS. The authors state that protein speciation is likely to increase the chance of proteins to be determined in 2-DE/MS, though admitting that low-sensitivity 2DE-MS methods were used in this study. In agreement with Kwiatkowski et al., we are convinced that the difference between 2DE-MS and bottom-up LC-MS will disappear, if high-resolution 2DE is combined with identification by a high-sensitivity LC-Orbitrap-MS. Meta-analysis of proteomic data is surely a promising tool, though the technological progress in 2DE and MS has to reach a plateau to enable useful comparisons.

Innovating the Concept and Practice of Two-Dimensional Gel Electrophoresis in the Analysis of Proteomes at the Proteoform Level.

Two-dimensional gel electrophoresis (2DE) is an important and well-established technical platform enabling extensive top-down proteomic analysis. However, the long-held but now largely outdated conventional concepts of 2DE have clearly impacted its application to in-depth investigations of proteomes at the level of protein species/proteoforms. It is time to popularize a new concept of 2DE for proteomics. With the development and enrichment of the proteome concept, any given "protein" is now recognized to consist of a series of proteoforms. Thus, it is the proteoform, rather than the canonical protein, that is the basic unit of a proteome, and each proteoform has a specific isoelectric point (pI) and relative mass (Mr ). Accordingly, using 2DE, each proteoform can routinely be resolved and arrayed according to its different pI and Mr . Each detectable spot contains multiple proteoforms derived from the same gene, as well as from different genes. Proteoforms derived from the same gene are distributed into different spots in a 2DE pattern. High-resolution 2DE is thus actually an initial level of separation to address proteome complexity and is effectively a pre-fractionation method prior to analysis using mass spectrometry (MS). Furthermore, stable isotope-labeled 2DE coupled with high-sensitivity liquid chromatography-tandem MS (LC-MS/MS) has tremendous potential for the large-scale detection, identification, and quantification of the proteoforms that constitute proteomes.

Small heat shock protein speciation: novel non-canonical 44 kDa HspB5-related protein species in rat and human tissues.

When analyzing small stress proteins of rat and human tissues by electrophoretic methods followed by western blotting, and using the anti-HspB1/anti-HspB5 antibody clone 8A7, we unexpectedly found a protein with a molecular mass of ~44 kDa. On two-dimensional gels, this protein resolved into four distinct species. Electrophoretic and immunological evidence suggests that this 44 kDa protein is a derivative of HspB5, most likely a covalently linked HspB5 dimer. This HspB5-like 44 kDa protein (HspB5L-P44) is particularly abundant in rat heart, brain, and renal cortex and glomeruli. HspB5L-P44 was also found in human brains, including those from patients with Alexander disease, a condition distinguished by cerebral accumulation of HspB5. Gray matter of such a patient contained an elevated amount of HspB5L-P44. A spatial model of structurally ordered dimeric HspB5 α-crystallin domains reveals the exposed and adjacent position of the two peptide segments homologous to the HspB1-derived 8A7 antigen determinant peptide (epitope). This explains the observed extraordinary high avidity of the 8A7 antibody towards HspB5L-P44, as opposed to commonly used HspB5-specific antibodies which recognize other epitopes. This scenario also explains the remarkable fact that no previous study reported the existence of HspB5L-P44 species. Exposure of rat endothelial cells to UV light, an oxidative stress condition, temporarily increased HspB5L-P44, suggesting physiological regulation of the dimerization. The existence of HspB5L-P44 supports the protein speciation discourse and fits to the concept of the protein code, according to which the expression of a given gene is reflected only by the complete set of the derived protein species.

How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?

Two-dimensional gel electrophoresis (2DE) in proteomics is traditionally assumed to contain only one or two proteins in each 2DE spot. However, 2DE resolution is being complemented by the rapid development of high sensitivity mass spectrometers. Here we compared MALDI-MS, LC-Q-TOF MS and LC-Orbitrap Velos MS for the identification of proteins within one spot. With LC-Orbitrap Velos MS each Coomassie Blue-stained 2DE spot contained an average of at least 42 and 63 proteins/spot in an analysis of a human glioblastoma proteome and a human pituitary adenoma proteome, respectively, if a single gel spot was analyzed. If a pool of three matched gel spots was analyzed this number further increased up to an average of 230 and 118 proteins/spot for glioblastoma and pituitary adenoma proteome, respectively. Multiple proteins per spot confirm the necessity of isotopic labeling in large-scale quantification of different protein species in a proteome. Furthermore, a protein abundance analysis revealed that most of the identified proteins in each analyzed 2DE spot were low-abundance proteins. Many proteins were present in several of the analyzed spots showing the ability of 2DE-MS to separate at the protein species level. Therefore, 2DE coupled with high-sensitivity LC-MS has a clearly higher sensitivity as expected until now to detect, identify and quantify low abundance proteins in a complex human proteome with an estimated resolution of about 500 000 protein species. This clearly exceeds the resolution power of bottom-up LC-MS investigations.

Sex differences in murine myocardium are not exclusively regulated by gonadal hormones.

We investigated sex differences in cardiac protein patterns of intact and castrated mice using proteomics and 1D and 2D immunoblotting. To exclude differences concerning developmental aspects gonadectomy was conducted in mature mice at the age of three months. The main sex-related regulation in the protein pattern of the myocardium occurred for proteins involved in metabolic processes whereas only few proteins involved in other pathways underwent a regulation. Many regulated proteins (2/3) displayed a characteristic V form, which means that these proteins are up- or down-regulated in sexually mature compared to young mice and are back-regulated after castration, emphasizing a direct regulation by gonadal hormones. Several other spots (1/3) showed the same male/female regulation or a drastic increase in male/female spot intensity ratio after castration, suggesting either a regulation independent of sex hormones or a removal of an inhibiting feedback mechanism by gonadectomy. Technically, we found that it cannot be expected that a single spot contains only one protein species and that one protein is present in only one spot. We thus propose for proteomic investigations to identify/quantify all spots of a 2-DE pattern to obtain information about protein speciation and its potential importance for function and pathology. BIOLOGICAL SIGNIFICANCE: Sex related differences in cardiovascular disease, including risk factors, disease manifestation and outcomes, are far from being well understood, and improved biological understanding of these differences in the healthy myocardium is of great importance. We investigated sex related changes of myocardial protein pattern in intact and castrated mice at different ages and found metabolic proteins to be highly regulated, some of which independently from gonadal hormones.

Analysis of T4SS-induced signaling by H. pylori using quantitative phosphoproteomics.

Helicobacter pylori is a Gram-negative bacterial pathogen colonizing the human stomach. Infection with H. pylori causes chronic inflammation of the gastric mucosa and may lead to peptic ulceration and/or gastric cancer. A major virulence determinant of H. pylori is the type IV secretion system (T4SS), which is used to inject the virulence factor CagA into the host cell, triggering a wide range of cellular signaling events. Here, we used a phosphoproteomic approach to investigate tyrosine signaling in response to host-pathogen interaction, using stable isotope labeling in cell culture (SILAC) of AGS cells to obtain a differential picture between multiple infection conditions. Cells were infected with wild type H. pylori P12, a P12Δ CagA deletion mutant, and a P12Δ PAI deletion mutant to compare signaling changes over time and in the absence of CagA or the T4SS. Tryptic peptides were enriched for tyrosine (Tyr) phosphopeptides and analyzed by nano-LC-Orbitrap MS. In total, 85 different phosphosites were found to be regulated following infection. The majority of phosphosites identified were kinases of the MAPK family. CagA and the T4SS were found to be key regulators of Tyr phosphosites. Our findings indicate that CagA primarily induces activation of ERK1 and integrin-linked factors, whereas the T4SS primarily modulates JNK and p38 activation.

The proteomics quantification dilemma.

Proteomics is dominated today by the protein expression discourse, which favorites the bottom-up approach because of its high throughput and its high sensitivity. For quantification this proceeding is misleading, if a protein is present with more than one protein species in the sample to be analyzed. The protein speciation discourse considers this more realistic situation and affords the top-down procedures or at least a separation of the protein species in advance to identification and quantification. Today all of the top-down procedures are one order of magnitude less sensitive than the bottom-up ones. To increase sensitivity and to increase throughput are major challenges for proteomics of the next years. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.

Analysis of protein species differentiation among mycobacterial low-Mr-secreted proteins by narrow pH range Immobiline gel 2-DE-MALDI-MS.

Secreted proteins of bacteria are preferentially capable of interacting with host cells and are therefore of special biological and medical interest. Narrow pH range 2-DE and MALDI-TOFTOF-MS combine high-resolution protein separation with highly sensitive identification of proteins. Secreted proteins of Mycobacterium tuberculosis were separated at the protein species level, distinguishing different protein species of one protein. We focused on the pI range 4.0-4.7 and the Mr range 6-20kDa of the 2-DE pattern. Out of 128 analyzed spots, 121 were identified resulting in 33 different proteins with 277 different protein species, accumulating in a mean of 8.4 protein species per protein. Overrepresentation was found for the protein classes "virulence, detoxification, adaption", "information pathways", "cell wall and cell processes" and "intermediary metabolism and respiration". Thus far, 15 protein species of the ESX-1 family are characterized with 100% sequence coverage. More automated 2-DE procedures and more sensitive identification techniques are required for complete characterization of all of the protein species even in highly enriched samples, such as culture filtrates. Only then the functional level of proteomics will be achieved and potential biomarkers can be postulated at the molecular level. BIOLOGICAL SIGNIFICANCE: Proteomics is dominated by bottom-up approaches largely ignoring protein speciation. A prerequisite to reach the protein species level is to obtain 100% sequence coverage, which is a major challenge in proteomics. Here we show complete sequence information with a 2-DE-MS approach for 15 protein species. Acetylation of the N-terminus of ESAT-6 inhibits interaction with CFP-10, with direct consequences for pathogen-host interaction. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Back to the future--the value of single protein species investigations.

In proteomics, in the past years, there was a focus on high throughput and reaching of large numbers of identified proteins with the basic discourse of protein expression. To avoid the impression of producing pure lists attempts are usually made to correlate proteins changed in amount between two biological situations to different pathways or protein interactions. This discourse is based on two simplifications, which limit the applicability of proteomics drastically: (i) it is sufficient to quantify a protein from several enzymatic digestion products; (ii) a biological situation is sufficiently described, if a peptide with its PTM is identified, resulting in long lists of modified peptides with data amounts, which are not anymore made accessible for the reader of a publication. The elucidation of N-terminal methylation of proteasome subunit Rpt1 in yeast by Kimura et al. (Proteomics 2013, 13, 3167-3174), which represents the focus on one protein, shows the value of solid chemical analysis with a complete data documentation and paves the way to proteomics based on the protein speciation discourse.

High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture(SILAC), two-dimensional gel electrophoresis(2DE) and nano-liquid chromatograpohy coupled to an LTQ-OrbitrapMass spectrometer.

The proteomics field has shifted over recent years from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gel-free workflows because of the tremendous developments in isotopic labeling techniques, nano-liquid chromatography, and high-resolution mass spectrometry. However, 2-DE still offers the highest resolution in protein separation. Therefore, we combined stable isotope labeling with amino acids in cell culture of controls and apoptotic HeLa cells with 2-DE and the subsequent analysis of tryptic peptides via nano-liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer to obtain quantitative data using the methods with the highest resolving power on all levels of the proteomics workflow. More than 1,200 proteins with more than 2,700 protein species were identified and quantified from 816 Coomassie Brilliant Blue G-250 stained 2-DE spots. About half of the proteins were identified and quantified only in single 2-DE spots. The majority of spots revealed one to five proteins; however, in one 2-DE spot, up to 23 proteins were identified. Only half of the 2-DE spots represented a dominant protein with more than 90% of the whole protein amount. Consequently, quantification based on staining intensities in 2-DE gels would in approximately half of the spots be imprecise, and minor components could not be quantified. These problems are circumvented by quantification using stable isotope labeling with amino acids in cell culture. Despite challenges, as shown in detail for lamin A/C and vimentin, the quantitative changes of protein species can be detected. The combination of 2-DE with high-resolution nano-liquid chromatography-mass spectrometry allowed us to identify proteomic changes in apoptotic cells that would be unobservable using any of the other previously employed proteomic workflows.

Dynein light chain 8a of Toxoplasma gondii, a unique conoid-localized ß-strand-swapped homodimer, is required for an efficient parasite growth.

Dynein light chain 8 (DLC8) is a ubiquitous eukaryotic protein regulating diverse cellular functions. We show that the obligate intracellular parasite Toxoplasma gondii harbors 4 DLC8 proteins (TgDLC8a-d), of which only TgDLC8a clusters in the mainstream LC8 class. TgDLC8b-d proteins form a divergent and alveolate-specific clade. TgDLC8b-d proteins are largely cytosolic, whereas TgDLC8a resides in the conoid at the apical end of T. gondii. The apical location of TgDLC8a is also not shared by its nearly identical Eimeria (EtDLC8a), Plasmodium (PfDLC8), or human (HsDLC8) orthologs. Notwithstanding an exclusive conoid targeting, TgDLC8a exhibits a classical LC8 structure. It forms a homodimer by swapping of the ß strands that interact with the antiparallel ß' strands of the opposing monomers. The TgDLC8a dimer contains two identical binding grooves and appears to be adapted for multitarget recognition. By contrast, the previously reported PfDLC8 homodimer is shaped by binding of the ß strand with the parallel ß' strand and lacks such a distinct binding interface. Our comparisons suggest an unexpected structural and functional divergence of the two otherwise conserved proteins from apicomplexan parasites. Finally, we demonstrate that a phosphomimetic S88E mutation renders the TgDLC8a-S88E mutant monomeric and cytosolic in T. gondii, and its overexpression inhibits the parasite growth in human fibroblasts.

Dietary phytoestrogen supplementation induces sex differences in the myocardial protein pattern of mice: a comparative proteomics study.

Elevated cardiovascular risk in postmenopausal women and beneficial actions of estrogen replacement in animal models have been related to protective effects of estrogens. However, randomized trials of hormone replacement therapy with synthetic estrogens in humans failed confirmation and phytoestrogens, natural plant hormones with agonistic properties for estrogen receptors, could represent potential alternatives. The aim of the present study is to characterize an animal model for alternative hormone replacement with genistein as a natural estrogenic compound. We performed a 2-DE/ESI-LC-MS approach in order to identify protein species varying with genistein receipt and sex in their relative abundance in the healthy murine heart (http://www.mpiib-berlin.mpg.de/2D-PAGE). Oral genistein treatment revealed a substantial effect on the relative abundance of both estrogen receptors. Several enzymes of the fatty acid metabolism and their transcriptional regulators varied differentially in male and in female animals, at the transcript and/or the protein species level. Increased levels of enzyme species involved in the oxidative phosphorylation and generation of ROS were accompanied by decreased amounts of antioxidants in male mice receiving genistein compared with control males, which have been previously associated with various pathological conditions. Exposure of female animals to genistein provoked an increased abundance of two species of LIM domain-binding protein and one species of desmin. These proteins have been associated with cardiac hypertrophy and our data warrant caution for the use of them as molecular markers, since the animals did not exhibit any histological signs of cardiac hypertrophy.

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.

Membrane-SPINE: an improved method to identify protein-protein interaction partners of membrane proteins in vivo.

Membrane proteins are crucial for many essential cellular processes. As membrane proteins function in complexes, methods to detect and to characterize membrane protein-protein interactions are undoubtedly required. Therefore, we developed the "Membrane-Strep-tagged protein interaction experiment" (Membrane-SPINE) that combines the specific purification of a Strep-tagged membrane protein with the reversible fixation of protein complexes by formaldehyde cross-linking. In combination with MS analysis, we suggest Membrane-SPINE as a powerful tool to identify unknown interaction partners of membrane proteins in vivo.

Towards the analysis of protein species: an overview about strategies and methods.

The deciphering of the relationship between function and exact chemical composition of a defined protein species in the context of the proteome is one of the major challenges in proteomics and molecular cell physiology. In the Special Issue of Amino Acids about the analysis of protein species current approaches are reviewed and new methods described focusing on the investigation of protein species. On the basis of the articles in this Special Issue it can be summarized that first important and promising steps towards the comprehensive analysis of protein species have been done. It is already possible to obtain full (100%) sequence coverage of proteins by mass spectrometry, if the amount of proteins available for their analysis allows their proteolytic degradation by more than one protease and the subsequent mass spectrometric analysis of the resulting peptides. Employing affinity chromatography helps to analyse proteins with defined post-translational modifications thus opening a targeted view on e.g. the phosphoproteome. In the future the aim to identify the exact chemical composition including not one but every posttranslational modification and complete sequence coverage on the protein species level should be achievable with further progress in sample preparation techniques, especially concerning separation techniques on the protein level, mass spectrometry and algorithms for mass spectrometric data processing. For determining the function of defined protein species a closer cooperation between cell biologists and proteomics experts is desirable.

Adaptation of proteomic techniques for the identification and characterization of protein species from murine heart.

Disturbed energy metabolism with impaired fatty acid oxidation, ATP synthesis and changing levels of contractile proteins has been observed during the development and manifestation of cardiovascular diseases, with the latter showing sexual differences in terms of onset, manifestation and progress. Estrogenic compounds, such as estrogens and phytoestrogens, are known to exert beneficial effects on several cardiovascular parameters. However, global studies implying the normal, non-failing myocardium are rare. Thus, identifying and characterizing protein patterns involved in the maintenance of normal heart physiology at the protein species level will help understanding disease conditions. In this study, we performed an adapted 2-DE/MS approach in order to identify and characterize post-translational modified and truncated protein species from murine heart. Female and male animals of different age were receiving the phytoestrogen genistein and comparative analyses were performed to identify sex and genistein treatment-related effects. Selected 2-DE spots that exposed varying abundance between animal groups and identified as identical proteins were subject to multi-protease cleavage to generate an elevated sequence coverage enabling characterization of post-translational modifications and truncation loci via high-resolution MS. Several truncated, phosphorylated and acetylated species were identified for mitochondrial ATP synthase, malate dehydrogenase and trifunctional enzyme subunit alpha. However, confirmation of several of these modifications by manual spectra interpretation failed. Thus, our results warrant caution for the blind trust in software output. For the regulatory light chain of myosin, we identified an N-terminal processed species, which so far has been related to ischemic conditions only. We tried to unravel the information content of protein species separated by high-resolution 2-DE as an alternative to high-throughput proteomics, which mainly is interested in lists of protein names, ignoring the protein species identity.

Quantitative proteome analysis of the 20S proteasome of apoptotic Jurkat T cells.

Regulated proteolysis plays important roles in cell biology and pathological conditions. A crosstalk exists between apoptosis and the ubiquitin-proteasome system, two pathways responsible for regulated proteolysis executed by different proteases. To investigate whether the apoptotic process also affects the 20S proteasome, we performed three independent SILAC-based quantitative proteome approaches: 1-DE/MALDI-MS, small 2-DE/MALDI-MS and large 2-DE/nano-LC-ESI-MS. Taking the results of all experiments together, no quantitative changes were observed for the α- and ß-subunits of the 20S proteasome except for subunit α7. This protein was identified in two protein spots with a down-regulation of the more acidic protein species (α7a) and up-regulation of the more basic protein species (α7b) during apoptosis. The difference in these two α7 protein species could be attributed to oxidation of cysteine-41 to cysteine sulfonic acid and phosphorylation at serine-250 near the C terminus in α7a, whereas these modifications were missing in α7b. These results pointed to the biological significance of posttranslational modifications of proteasome subunit α7 after induction of apoptosis.