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.

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.

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 However, the long-held but now largely outdated conventional concepts of 2DE have clearly impacted its application to in-depth investigations of proteomes at the level of protein species/proteoforms.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.

Nuclear cGAS suppresses DNA repair and promotes tumorigenesis.

Accurate repair of DNA double-stranded breaks by homologous recombination preserves genome integrity and inhibits tumorigenesis. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that activates innate immunity by initiating the STING-IRF3-type I IFN signalling cascade. Recognition of ruptured micronuclei by cGAS links genome instability to the innate immune response, but the potential involvement of cGAS in DNA repair remains unknown.

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.

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

Unlabelled: 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.

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.

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.

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.

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.

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

Unlabelled: 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.

Dynein light chain 8a of Toxoplasma gondii, a unique conoid-localized β-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.

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.

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.

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.

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.

Helicobacter pylori HP0518 affects flagellin glycosylation to alter bacterial motility.

Helicobacter pylori is a human gastric pathogen associated with gastric and duodenal ulcers as well as gastric cancer. Mounting evidence suggests this pathogen's motility is prerequisite for successful colonization of human gastric tissues. Here, we isolated an H.

Influence of RET/PTC1 and RET/PTC3 oncoproteins in radiation-induced papillary thyroid carcinomas on amounts of cytoskeletal protein species.

Radiation-induced human papillary thyroid carcinomas (PTCs) show a high prevalence of fusions of the RET proto-oncogene to heterologous genes H4 (RET/PTC1) and ELE1 (RET/PTC3), respectively. In contrast to the normal membrane-bound RET protein, aberrant RET fusion proteins are constitutively active oncogenic cytosolic proteins that can lead to malignant transformation of thyroid epithelia. To detect specific tumor-associated protein changes that reflect the effect of RET/PTC fusion proteins, we analyzed normal thyroid tissues, thyroid tumors of the RET/PTC1 and RET/PTC3 type and their respective lymph node metastases by a combination of high-resolution two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-mass spectrometry.

Proteomic identification of secreted proteins of Propionibacterium acnes.

Background: The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce.

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.

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.

Helicobacter pylori proteomics by 2-DE/MS, 1-DE-LC/MS and functional data mining.

With its predicted proteome of 1550 proteins (data set Etalon) Helicobacter pylori 26695 represents a perfect model system of medium complexity for investigating basic questions in proteomics. We analyzed urea-solubilized proteins by 2-DE/MS (data set 2-DE) and by 1-DE-LC/MS (Supprot); proteins insoluble in 9 M urea but solubilized by SDS (Pellet); proteins precipitating in the Sephadex layer at the application side of IEF (Sephadex) by 1-DE-LC/MS; and proteins precipitating close to the application side within the IEF gel by LC/MS (Startline). The experimental proteomics data of H.