Variation in supramolecular organisation of the photosynthetic membrane of Rhodobacter sphaeroides induced by alteration of PufX

In purple bacteria of the genus Rhodobacter (Rba.), an LH1 antenna complex surrounds the photochemical reaction centre (RC) with a PufX protein preventing the LH1 complex from completely encircling the RC. In membranes of Rba. sphaeroides, RC–LH1 complexes associate as dimers which in turn assemble into longer range ordered arrays. The present work uses linear dichroism (LD) and dark-minus-light difference LD (ΔLD) to probe the organisation of genetically altered RC–LH1 complexes in intact membranes. The data support previous proposals that Rba. capsulatus, and Rba. sphaeroides heterologously expressing the PufX protein from Rba. capsulatus, produce monomeric core complexes in membranes that lack long-range order. Similarly, Rba. sphaeroides with a point mutation in the Gly 51 residue of PufX, which is located on the membrane-periplasm interface, assembles mainly non-ordered RC–LH1 complexes that are most likely monomeric. All the Rba. sphaeroides membranes in their ΔLD spectra exhibited a spectral fingerprint of small degree of organisation implying the possibility of ordering influence of LH1, and leading to an important conclusion that PufX itself has no influence on ordering RC–LH1 complexes, as long-range order appears to be induced only through its role of configuring RC–LH1 complexes into dimers.     

Analytical Utility of Mass Spectral Binning in Proteomic Experiments by SPectral Immonium Ion Detection (SPIID)

Unambiguous identification of tandem mass spectra is a cornerstone in mass-spectrometry-based proteomics. As the study of post-translational modifications (PTMs) by means of shotgun proteomics progresses in depth and coverage, the ability to correctly identify PTM-bearing peptides is essential, increasing the demand for advanced data interpretation. Several PTMs are known to generate unique fragment ions during tandem mass spectrometry, the so-called diagnostic ions, which unequivocally identify a given mass spectrum as related to a specific PTM. Although such ions offer tremendous analytical advantages, algorithms to decipher MS/MS spectra for the presence of diagnostic ions in an unbiased manner are currently lacking. Here, we present a systematic spectral-pattern-based approach for the discovery of diagnostic ions and new fragmentation mechanisms in shotgun proteomics datasets. The developed software tool is designed to analyze large sets of high-resolution peptide fragmentation spectra independent of the fragmentation method, instrument type, or protease employed. To benchmark the software tool, we analyzed large higher-energy collisional activation dissociation datasets of samples containing phosphorylation, ubiquitylation, SUMOylation, formylation, and lysine acetylation. Using the developed software tool, we were able to identify known diagnostic ions by comparing histograms of modified and unmodified peptide spectra. Because the investigated tandem mass spectra data were acquired with high mass accuracy, unambiguous interpretation and determination of the chemical composition for the majority of detected fragment ions was feasible. Collectively we present a freely available software tool that allows for comprehensive and automatic analysis of analogous product ions in tandem mass spectra and systematic mapping of fragmentation mechanisms related to common amino acids.In mass spectrometry (MS)-based proteomics, protein mixtures are digested into peptides using standard proteases such as trypsin or Lys-C (1). The complex peptide mixture is separated via liquid chromatography (LC) directly coupled to MS, and the eluting peptide ions are electrosprayed into the vacuum of the mass spectrometer, where a peptide mass spectrum is recorded (2). In the mass spectrometer, selected peptide ions are fragmented, most commonly through the collision of peptide molecular ions with inert gas molecules in a technique referred to as either collision-induced dissociation (CID)¹ or collisionally activated dissociation (3, 4). During this energetic collision, some of the deposited kinetic energy is converted into internal energy, which results in peptide bond breakage and fragmentation of the molecular peptide ion into sequence-specific ions (5). Identification of the analyzed peptide is then performed by scanning the measured peptide mass and list of fragment masses against a protein sequence database (6). Overall this approach provides a rapid and sensitive means of determining the primary sequence of peptides.During the fragmentation step, various types of fragment ions can be observed in the MS/MS spectrum. Their occurrence depends on the primary sequence of the investigated peptide, the amount of internal energy deposited, how the energy was introduced, the charge state, and other factors (7). Low-energy dissociation conditions as observed in ion trap CID mainly generate fragment ions containing sequence-specific amino acid information about the investigated peptides (8). This occurs because the energy deposited during this fragmentation method primarily facilitates the fragmentation of precursor ions yielding single peptide bond fragmentation between individual amino acids (9).With faster activation methods, such as beam-type/quadrupole CID (10), generated fragments can undergo further collisions. Multiple bonds can thereby be fragmented, giving rise to internal sequence ions, which in combination with regular b- and y-type cleavage produce specific amino-immonium ions (11). These immonium ions appear in the very low m/z range of the MS/MS spectrum, and for the majority of naturally occurring amino acids such immonium ions are unique for that particular residue (12, 13). Exceptions for this are the leucine/isoleucine and lysine/glutamine pairs, which produce immonium ions with the same chemical mass. Overall, immonium ions can confirm the presence of certain amino acid residues in a peptide, whereas information regarding the position or the stoichiometry of these amino acid residues cannot be ascertained. Because tryptic peptides on average contain 9 to 12 amino acids, they frequently contain many different residues; as a result, the analytical information hidden in the regular amino acid immonium ions might be limited. However, immonium ions can be used to support peptide sequence assignment during proteomic database searching (14).Contrary to the 20 naturally occurring residues, many amino acids can be modified by various post-translational modifications (PTMs), and these PTM-bearing residues can themselves generate unique immonium ions—the so-called diagnostic ions. The two most prominent examples are phosphorylation of tyrosine and acetylation of lysine residues (15), which generate diagnostic ions at m/z = 216.0424 and m/z = 126.0917, respectively. Thus, the presence of these unique ions in a MS/MS spectrum can unequivocally identify the sequenced peptide as harboring a given PTM. Evidently, knowledge regarding modification-specific diagnostic ions is of great importance for the identification and validation of modified peptides in MS-based proteomics (16, 17). Additionally, such PTM-specific information can be informative in targeted proteomics approaches facilitating MS/MS precursor ion scanning (18) and become valuable in post-acquisition analysis involving extracted ion chromatograms for specific m/z values. Moreover, information regarding diagnostic ions can be a powerful addition to analytical approaches such as selected reaction monitoring, a targeted technique that relies on ion-filtering capabilities to comprehensively study peptides and PTMs (19).Currently only a minor subset of modified amino acids has been investigated for diagnostic ions, primarily because of the lack of unbiased methods for mapping such ions in large-scale proteomics experiments. The identification of diagnostic ions is a labor-intensive endeavor, requiring manual interpretation of large numbers of MS/MS spectra for proper validation of low-mass fragmentation ions. As a result, most studies on diagnostic ions have been performed on a few selected synthetic peptides, as the interrogation of larger biological datasets has not been feasible (15, 20).Here we describe a proteomic approach utilizing a novel algorithm based upon binning of tandem mass spectra for fast and automated mapping of analogously occurring product ions. The developed algorithm is completely independent of instrument type and fragmentation technique employed, but it performs more favorably under experimental conditions that augment the generation of immonium ions. As a result, the performance of the algorithm is benchmarked on data derived from LTQ Orbitrap Velos and Q Exactive mass spectrometers, which exhibit improved HCD performance (21–23). HCD has proven to be a powerful fragmentation technique, particularly for PTM analysis (24, 25), as no low mass detection cutoff is observed as compared with fragmentation experiments on ion trap mass spectrometers (26). Moreover, the beam-type energy deposited during HCD fragmentation allows for improved generation of both immonium and other sequence-related ions relative to CID (27, 28). Additionally, HCD experiments are performed at very high resolution, yielding high mass accuracy (<10 ppm) on all detected fragment ions, which allows the algorithm to utilize very narrow mass binning and hence easily determine the exact chemical composition of any novel detected ions.Briefly, the algorithm takes all significantly identified MS/MS spectra and bins them together in discrete mass bins. As commonly occurring ions, such as immonium and diagnostic ions, will have same chemical composition and consequently the same m/z, they will cluster in the same mass bins, whereas sequence-specific fragment ions will scatter across the binned mass range. For validation of the presented approach, we mapped known and novel diagnostic ions from a variety of PTM-bearing amino acids, demonstrating the sensitivity and specificity of the method. Moreover, we demonstrate that mass spectral binning additionally can be employed for automated mapping of composition-specific neutral losses from large-scale proteomic experiments.     

Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro

Cotranslational protein maturation is often studied in cell-free translation mixtures, using stalled ribosome-nascent chain complexes produced by translating truncated mRNA. This approach has two limitations: (i) it can be technically challenging, and (ii) it only works in vitro, where the concentrations of cellular components differ from concentrations in vivo. We have developed a method to produce stalled ribosomes bearing nascent chains of a specified length by using a 'stall sequence', derived from the Escherichia coli SecM protein, which interacts with residues in the ribosomal exit tunnel to stall SecM translation. When the stall sequence is expressed at the end of nascent chains, stable translation-arrested ribosome complexes accumulate in intact cells or cell-free extracts. SecM-directed stalling is efficient, with negligible effects on viability. This method is straightforward and suitable for producing stalled ribosome complexes in vivo, permitting study of the length-dependent maturation of nascent chains in the cellular milieu.     

The phosphotyrosine peptide binding specificity of Nck1 and Nck2 Src homology 2 domains

Nck proteins are essential Src homology (SH) 2 and SH3 domain-bearing adapters that modulate actin cytoskeleton dynamics by linking proline-rich effector molecules to tyrosine kinases or phosphorylated signaling intermediates. Two mammalian pathogens, enteropathogenic Escherichia coli and vaccinia virus, exploit Nck as part of their infection strategy. Conflicting data indicate potential differences in the recognition specificities of the SH2 domains of the isoproteins Nck1 (Nckalpha) and Nck2 (Nckbeta and Grb4). We have characterized the binding specificities of both SH2 domains and find them to be essentially indistinguishable. Crystal structures of both domains in complex with phosphopeptides derived from the enteropathogenic E. coli protein Tir concur in identifying highly conserved, specific recognition of the phosphopeptide. Differential peptide recognition can therefore not account for the preference of either Nck in particular signaling pathways. Binding studies using sequentially mutated, high affinity phosphopeptides establish the sequence variability tolerated in peptide recognition. Based on this binding motif, we identify potential new binding partners of Nck1 and Nck2 and confirm this experimentally for the Arf-GAP GIT1.     

A blood based 12-miRNA signature of Alzheimer disease patients

Background

Alzheimer disease (AD) is the most common form of dementia but the identification of reliable, early and non-invasive biomarkers remains a major challenge. We present a novel miRNA-based signature for detecting AD from blood samples.

Results

We apply next-generation sequencing to miRNAs from blood samples of 48 AD patients and 22 unaffected controls, yielding a total of 140 unique mature miRNAs with significantly changed expression levels. Of these, 82 have higher and 58 have lower abundance in AD patient samples. We selected a panel of 12 miRNAs for an RT-qPCR analysis on a larger cohort of 202 samples, comprising not only AD patients and healthy controls but also patients with other CNS illnesses. These included mild cognitive impairment, which is assumed to represent a transitional period before the development of AD, as well as multiple sclerosis, Parkinson disease, major depression, bipolar disorder and schizophrenia. miRNA target enrichment analysis of the selected 12 miRNAs indicates an involvement of miRNAs in nervous system development, neuron projection, neuron projection development and neuron projection morphogenesis. Using this 12-miRNA signature, we differentiate between AD and controls with an accuracy of 93%, a specificity of 95% and a sensitivity of 92%. The differentiation of AD from other neurological diseases is possible with accuracies between 74% and 78%. The differentiation of the other CNS disorders from controls yields even higher accuracies.

Conclusions

The data indicate that deregulated miRNAs in blood might be used as biomarkers in the diagnosis of AD or other neurological diseases.     

Absorption and CD spectroscopy and modeling of various LH2 complexes from purple bacteria

The absorption (OD) and circular dichroism (CD) spectra of LH2 complexes from various purple bacteria have been measured and modeled. Based on the lineshapes of the spectra we can sort the LH2 complexes into two distinguishable groups: "acidophila"-like (type 1) and "molischianum"-like (type 2). Starting from the known geometric structures of Rhodopseudomonas (Rps.) acidophila and Rhodospirillum (Rsp.) molischianum we can model the OD and CD spectra of all species by just slightly varying some key parameters: the interaction strength, the energy difference of alpha- and beta-bound B850 bacteriochlorophylls (BChls), the orientation of the B800 and B850 BChls, and the (in)homogeneous broadening. Although the ring size can vary, the data are consistent with all the LH2 complexes having basically very similar structures.     

Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos

Over the past decade peptide sequencing by collision induced dissociation (CID) has become the method of choice in mass spectrometry-based proteomics. The development of alternative fragmentation techniques such as electron transfer dissociation (ETD) has extended the possibilities within tandem mass spectrometry. Recent advances in instrumentation allow peptide fragment ions to be detected with high speed and sensitivity (e.g., in a 2D or 3D ion trap) or at high resolution and high mass accuracy (e.g., an Orbitrap or a ToF). Here, we describe a comprehensive experimental comparison of using ETD, ion-trap CID, and beam type CID (HCD) in combination with either linear ion trap or Orbitrap readout for the large-scale analysis of tryptic peptides. We investigate which combination of fragmentation technique and mass analyzer provides the best performance for the analysis of distinct peptide populations such as N-acetylated, phosphorylated, and tryptic peptides with up to two missed cleavages. We found that HCD provides more peptide identifications than CID and ETD for doubly charged peptides. In terms of Mascot score, ETD FT outperforms the other techniques for peptides with charge states higher than 2. Our data shows that there is a trade-off between spectral quality and speed when using the Orbitrap for fragment ion detection. We conclude that a decision-tree regulated combination of higher-energy collisional dissociation (HCD) and ETD can improve the average Mascot score.