Bottom-up Sequence Research Articles

Coacervate-Assisted Polymerization-Induced Self-Assembly of Chiral Alternating Copolymers into Hierarchical Bishell Capsules with Sub-5nm Ultrathin Lamellae.

Hierarchical self-assembly of synthetic polymers in solution represents one of the sophisticated strategies to replicate the natural superstructures which lay the basis for their superb functions. However, it is still quite challenging to increase the degree of complexity of the as-prepared assemblies, especially in a large scale. Liquid-liquid phase separation (LLPS) widely exists in cells and is assumed to be responsible for the formation of many cellular organelles without membranes. Herein, through integrating LLPS with the polymerization-induced self-assembly (PISA), a coacervate-assisted PISA (CAPISA) methodology to realize the one-pot and scalable preparation of hierarchical bishell capsules (BCs) from nanosheets with ultrathin lamellae phase (sub-5nm), microflakes, unishell capsules to final BCs in a bottom-up sequence is presented. Both the self-assembled structure and the dynamic formation process of BCs have been disclosed. Since CAPISA has combined the advantages of coacervates, click chemistry, interfacial reaction and PISA, it is believed that it will become a promising option to fabricate biomimetic polymer materials with higher structural complexity and more sophisticated functions.

Identification of Novel Microcystins Using High-Resolution MS and MSn with Python Code.

Cyanotoxins called microcystins (MCs) are highly toxic and can be present in drinking water sources. Determining the structure of MCs is paramount because of its effect on toxicity. Though over 300 MC congeners have been discovered, many remain unidentified. Herein, a method is described for the putative identification of MCs using liquid chromatography (LC) coupled with high-resolution (HR) Orbitrap mass spectrometry (MS) and a new bottom-up sequencing strategy. Maumee River water samples were collected during a harmful algal bloom and analyzed by LC-MS with simultaneous HRMS and MS/MS. Unidentified ions with characteristic MC fragments (135 and 213 m/z) were recognized as possible novel MC congeners. An innovative workflow was developed for the putative identification of these ions. Python code was written to generate the potential structures of unidentified MCs and to assign ions after the fragmentation for structural confirmation. The workflow enabled the putative identification of eight previously reported MCs for which standards are not available and two newly discovered congeners, MC-HarR and MC-E(OMe)R.

Strategic sill pillar design for reduced hanging wall overbreak in longhole mining

Steeply dipping, vein and tabular orebodies are traditionally extracted with longitudinal retreat mining methods such as Eureka and Avoca in a bottom-up sequence with delayed backfill. To increase productivity, sill pillars in the orebody are used to separate mining zones thus allowing production to take place simultaneously in two or more zones. While such mining methods are productive, they may be accompanied with high volumes of hanging wall overbreak causing significant unplanned ore dilution. In this work, it is shown through a mine design case study of a narrow vein deposit that a sill pillar could also play a significant role in limiting hanging wall overbreak. To demonstrate the role of sill pillar, a novel numerical modelling scheme is proposed to account for progressive stope wall overbreak. A numerical modelling approach of element death and rebirth is developed to allow for the detected stope overbreak to be immediately removed and replaced with backfill material before upper-level stope extraction. It is further shown that the average overbreak volume could be reduced by as much as 33% when the sill pillar is strategically placed in the lower half of a mine plan.

Exploring the molecular content of CHO exosomes during bioprocessing

In biopharmaceutical production, Chinese hamster ovary (CHO) cells derived from Cricetulus griseus remain the most commonly used host cell for recombinant protein production, especially antibodies. Over the last decade, in-depth multi-omics characterization of these CHO cells provided data for extensive cell line engineering and corresponding increases in productivity. However, exosomes, extracellular vesicles containing proteins and nucleic acids, are barely researched at all in CHO cells. Exosomes have been proven to be a ubiquitous mediator of intercellular communication and are proposed as new biopharmaceutical format for drug delivery, indicator reflecting host cell condition and anti-apoptotic factor in spent media. Here we provide a brief overview of different separation techniques and subsequently perform a proteome and regulatory, non-coding RNA analysis of exosomes, derived from lab-scale bioreactor cultivations of a CHO-K1 cell line, to lay out reference data for further research in the field. Applying bottom-up orbitrap shotgun proteomics and next-generation small RNA sequencing, we detected 1395 proteins, 144 micro RNA (miRNA), and 914 PIWI-interacting RNA (piRNA) species differentially across the phases of a batch cultivation process. The exosomal proteome and RNA data are compared with other extracellular fractions and cell lysate, yielding several significantly exosome-enriched species.Graphical Key points• First-time comprehensive protein and miRNA characterization of CHO exosomes.• Isolation protocol and time point of bioprocess strongly affect quality of extracellular vesicles.• CHO-derived exosomes also contain numerous piRNA species of yet unknown function.

Behaviour of an underground metro station for different constitutive models - A study on the influence of Mohr-Coulomb and other advanced constitutive models using PLAXIS 2D for the proposed Chennai Metro Rail Phase 2 project

The Phase 2 of the proposed Chennai Metro Rail Project contains underground stations of depths below ground varying from 20m for a 2 level station to 32m for a 4 level station. Diaphragm walls have been chosen for the purpose of earth retention during construction stage and also to be a part of the permanent structure. The centre part of the stations is constructed using top-down methodology and the ends of the station which are used for the purpose of TBM launching and retrieval activities are constructed using either bottom-up or top-down methodologies. The behaviour of the part of the station constructed using top down sequence will be different from the part constructed using bottom up sequence. The current study focusses on the comparison of the influence of the various constitutive models available in the FEM software package PLAXIS 2D such as the basic Mohr-Coulomb model and advanced models such as Hardening soil model and Hardening soil small strain stiffness (HSS) models on the bending moments, shear forces of the diaphragm walls, ground deformations, strut forces etc. This comparative study has been performed on both the top-down and bottom-up sequences and for the different ground conditions along the proposed second phase metro rail alignment in Chennai region of India.

Optimal Collision Energies and Bioinformatics Tools for Efficient Bottom-up Sequence Validation of Monoclonal Antibodies.

Rigorous validation of amino acid sequence is fundamental in the characterization of original and biosimilar protein biopharmaceuticals. Widely accepted workflows are based on bottom-up mass spectrometry, and they often require multiple techniques and significant manual work. Here, we demonstrate that optimization of a set of tandem mass spectroscopy (MS/MS) collision energies and automated combination of all available information in the measurements can increase the sequence validated by one technique close to the inherent limits. We created a software (called "Serac") that consumes results of the Mascot database search engine and identifies the amino acids validated by bottom-up MS/MS experiments using the most rigorous, industrially acceptable definition of sequence coverage (we term this "confirmed sequence coverage"). The software can combine spectra at the level of amino acids or fragment ions to exploit complementarity, provides full transparency to justify validation, and reduces manual effort. With its help, we investigated collision energy dependence of confirmed sequence coverage of individual peptides and full proteins on trypsin-digested monoclonal antibody samples (rituximab and trastuzumab). We found the energy dependence to be modest, but we demonstrated the benefit of using spectra taken at multiple energies. We describe a workflow based on 2-3 LC-MS/MS runs, carefully selected collision energies, and a fragment ion level combination, which yields ∼85% confirmed sequence coverage, 25%-30% above that from a basic proteomics protocol. Further increase can mainly be expected from alternative digestion enzymes or fragmentation techniques, which can be seamlessly integrated to the processing, thereby allowing effortless validation of full sequences.

Primary and Higher Order Structure of the Reaction Center from the Purple Phototrophic Bacterium Blastochloris viridis: A Test for Native Mass Spectrometry.

The reaction center (RC) from the phototrophic bacterium Blastochloris viridis was the first integral membrane protein complex to have its structure determined by X-ray crystallography and has been studied extensively since then. It is composed of four protein subunits, H, M, L, and C, as well as cofactors, including bacteriopheophytin (BPh), bacteriochlorophyll (BCh), menaquinone, ubiquinone, heme, carotenoid, and Fe. In this study, we utilized mass spectrometry-based proteomics to study this protein complex via bottom-up sequencing, intact protein mass analysis, and native MS ligand-binding analysis. Its primary structure shows a series of mutations, including an unusual alteration and extension on the C-terminus of the M-subunit. In terms of quaternary structure, proteins such as this containing many cofactors serve to test the ability to introduce native-state protein assemblies into the gas phase because the cofactors will not be retained if the quaternary structure is seriously perturbed. Furthermore, this specific RC, under native MS, exhibits a strong ability not only to bind the special pair but also to preserve the two peripheral BCh's.

Protein de novo sequencing by top-down and middle-down MS/MS: Limitations imposed by mass measurement accuracy and gaps in sequence coverage

With the increasing accessibility of Fourier transform (FT) mass spectrometers, top-down/middle-down MS/MS characterization of protein sequences is rapidly gaining popularity. Compared to conventional bottom-up sequencing, the top-down/middle-down approach offers the advantages of fast sample preparation and unambiguous characterization of proteoforms in a mixture. If the modified or mutated peptide segment of interest is not found or recovered with the bottom-up approaches, top-down becomes attractive relative to spending time seeking the right enzyme or chromatographic approach. Here, we discuss the potential and limitations of protein sequence analysis by top-down/middle-down MS/MS alone. Even if 100% protein sequence coverage is achieved by MS/MS, fragment mass error tolerance as low as 1 ppm or 0.5 ppm is needed to differentiate glutamine from lysine at positions not exceeding 330 amino acids (AAs) or 660 AAs from the N-/C-terminus for a protein with 660 AAs (72,760 Da) or 1320 AAs (145,520 Da). To characterize the “AA sequence gap” between two adjacent fragments, we show that the number of gap AA sequences with identical masses for di-, tri-, and tetra-AA gaps grows exponentially with increasing number of gap amino acids. If peptide fragment mass could be measured exactly (in practice, to 0.00001 Da), it would then be possible to define the overall atomic composition for the group of amino acids spanning a product ion gap 3–4 amino acids long. However, when we consider any 3–4 amino acid gap, we find that 50–75% of the possible compositions describe at least two sets of amino acids. Moreover, a next-generation protein fragment deconvolution algorithm is critical to exploit the experimentally observed high mass accuracy generated from the 21 T FT-ICR MS/MS for high confidence and high throughput top-down/middle-down analysis of proteins with unknown sequences. Finally, we show that de novo top-down/middle-down MS/MS can determine the germline sequence category for a given monoclonal antibody (mAb) and further serve to identify its novel mutations.

Sequencing of superficial trunk muscle activation during range-of-motion tasks

Altered lumbo-pelvic activation sequences have been identified in individuals with low back pain. However, an analysis of activation sequences within different levels of the trunk musculature has yet to be conducted. This study identified the activation sequences characteristic of the trunk musculature during upright standing and range-of-motion tasks. Surface electromyography was recorded for eight trunk muscles bilaterally during trunk range-of-motion movement tasks in 30 participants. Cross-correlation was performed on 48 pairings of muscles, consisting of one lower- and one mid-level muscle, or one mid-level and one upper muscle. Time lags of the maximum cross-correlation coefficient were extracted and defined as a top-down or bottom-up activation sequence, or similar activation timing. Pairings that demonstrated a specific activation sequence in 50% or more of participants were then identified. Similar activation timing was consistently identified between muscle pairings for upright standing. Top-down sequences and similar timing were identified for abdominal – mid-level pairings in maximum flexion and slumped standing, respectively, while both tasks were characterized by bottom-up sequences when considering the lumbar and lower-thoracic erector spinae. Sequences were more variable across muscle pairings for lateral bend and axial twist tasks. These results provide insight into the synergy of the trunk musculature for movements in the three planes of motion. These findings can be used for comparison to low back pain populations, as altered activation sequences in these individuals may contribute to maladaptive loading patterns and consequently the development or exacerbation of low back pain.

Development of an Innovative Tool to Assess Student Physical Therapists’ Clinical Reasoning Competency

BACKGROUND AND PURPOSEIn the recent decades, the role of physical therapists (PTs) has been changing with an ever-increasing autonomy,1 accountability,2,3 and public expectation of professional competency.4 Since clinical reasoning is a vital component of professional competency,5 the American Physical Therapy Association has specified clinical reasoning as an educational outcome for graduates of physical therapist education programs.6 The importance of clinical reasoning in physical therapist education demands discipline-specific assessment tools7 and a thorough assessment system that incorporates different types of assessments.8Miller9 proposed a pyramidal framework, which divides clinical assessment into 4 hierarchical levels-knows, knows how, shows how, and does-in a bottom-up sequence. Rethans et al10 redefined the last 2 levels and labeled them as competency and performance, respectively. Miller's9 pyramid with the modified definitions provides a theoretical framework to form a comprehensive system for assessing the clinical reasoning of student physical therapists (SPTs). Figure 1 illustrates the assessment system, including the definition and testing environment of each level. Paper-based examinations are means to assess cognitive knowledge11 and can be used to test students' clinical reasoning at the bottom 2 levels. The Physical Therapist Clinical Performance Instrument: version 200612 (PT CPI: version 2006), on the other hand, serves as a tool to assess students' clinical reasoning at the performance level. To date, no tool has been designed to assess the competency level-what an individual is capable of doing in a controlled representation of professional practice.10 The lack of competency assessment makes the assessment system incomplete.Standardized patients have been used in examinations to provide simulated clinical encounters,9 which is a requirement for testing competency.10 For students who do not perform well on standardized patient examinations, their underlying problems have been categorized into 3 types: technique, cognitive, and noncognitive, with clinical reasoning and knowledge deficits identified as the cognitive problems.13 Identifying students who struggle with standardized patient examinations due to deficits in clinical reasoning can be a challenge, because clinical reasoning is a nearly invisible process14 that is not easily accessible or assessable. Therefore, the invisibility issue of clinical reasoning needs to be addressed in order to make standardized patient examinations a more suitable testing environment to assess students' clinical reasoning competency. Qualitative researchers adopted a method when studying the mostly invisible clinical reasoning process.15-17 The method required the research participants to encounter with clinical cases and verbalize their thinking underpinning the encounter, either concurrently (during the encounter) or retrospectively (after the encounter). Ericsson and Simon18 supported the use of verbal reports, particularly the concurrent and immediate retrospective ones, as a valid source of information about cognitive processes. Thus, combining the think aloud method with a standardized patient examination is theoretically sound in making the mostly invisible clinical reasoning visible and accurately represented in a simulated testing environment. Despite the theoretical soundness, scarce evidence is available pertaining to the combination. Pintz19 developed a standardized patient examination for assessing nurse practitioner students' diagnostic reasoning. Each student was required to work with a standardized patient alone, with the performance videorecorded for grading. The researcher reported occasions that the standardized patient had to prompt the students to think aloud. However, there was no description that the standardized patients were trained to do so. The lack of training might have made the prompting inconsistent. …

Research of Artificial Sites Green Roof for Energy-Saving Efficiency in Taiwan Taichung

In this study, set the empirical plane pilot area planting the groove (long 425cm × width 300cm × Height 20cm ) in the Taichung City residential area on the second floor roof, planting the groove bottom-up sequence laying frame high-rise, water proof layer, drainage layer,filter layer (non-woven, water-pottery-stone), the medium layer and planting of layer, planting the groove around the hollow brick interval income side to lighten the load. Noon hours exposed roof temperature of 25.49-37.56°C, green roofs surface only at 16.45-24.69°C, 17.74-24.79°C indoor temperature on the second floor, effectively prevent the roof surface temperature rise, and thus reduce the interior roof of the second temperature7.75-12.77°C. Building green roofs to cooling, mainly to reduce the building temperature, reducing air conditioning electricity consumption , accomplish cooling and energy-saving benefits.Compare different annual electricity consumption in the same period, extensive green roof to total electricity consumption of 860 K.Watts/hours , exposed roof to total electricity consumption in 1767 K.Watts/hours, 48.67% of the electricity consumption savings, to achieve energy efficiency.

A multi-scale strategy for discovery of novel endogenous neuropeptides in the crustacean nervous system

The conventional mass spectrometry (MS)-based strategy is often inadequate for the comprehensive characterization of various size neuropeptides without the assistance of genomic information. This study evaluated sequence coverage of different size neuropeptides in two crustacean species, blue crab Callinectes sapidus and Jonah crab Cancer borealis using conventional MS methodologies and revealed limitations to mid- and large-size peptide analysis. Herein we attempt to establish a multi-scale strategy for simultaneous and confident sequence elucidation of various sizes of peptides in the crustacean nervous system. Nine novel neuropeptides spanning a wide range of molecular weights (0.9-8.2kDa) were fully sequenced from a major neuroendocrine organ, the sinus gland of the spiny lobster Panulirus interruptus. These novel neuropeptides included seven allatostatin (A- and B-type) peptides, one crustacean hyperglycemic hormone precursor-related peptide, and one crustacean hyperglycemic hormone. Highly accurate multi-scale characterization of a collection of varied size neuropeptides was achieved by integrating traditional data-dependent tandem MS, improved bottom-up sequencing, multiple fragmentation technique-enabled top-down sequencing, chemical derivatization, and in silico homology search. Collectively, the ability to characterize a neuropeptidome with vastly differing molecule sizes from a neural tissue extract could find great utility in unraveling complex signaling peptide mixtures employed by other biological systems. Mass spectrometry (MS)-based neuropeptidomics aims to completely characterize the neuropeptides in a target organism as an important first step toward a better understanding of the structure and function of these complex signaling molecules. Although liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with data-dependent acquisition is a powerful tool in peptidomic research, it often lacks the capability for de novo sequencing of mid-size and large peptides due to inefficient fragmentation of peptides larger than 4kDa. This study describes a multi-scale strategy for complete and confident sequence elucidation of various sizes of neuropeptides in the crustacean nervous system. The aim is to fill a technical gap where the conventional strategy is inefficient for comprehensive characterization of a complex neuropeptidome without assistance of genomic information. Nine novel neuropeptides in a wide range of molecular weights (0.9-8.2kDa) were fully sequenced from a major neuroendocrine organ of the spiny lobster, P. interruptus. The resulting molecular information extracted from such multi-scale peptidomic analysis will greatly accelerate functional studies of these novel neuropeptides.

On a nanoscopically-informed shell theory of single-wall carbon nanotubes

This paper proposes a bottom-up sequence of modeling steps leading to a nanoscopically informed macroscopic theory of single-walled carbon nanotubes (SWCNTs). We provide a description of the geometry and the mechanics of the two most representative types of SWCNTs, armchair (A-) and zigzag (Z-), of their modules and of their elementary bond units. We believe ours to be the simplest shell theory that accounts accurately for the linearly elastic response of both A- and Z-CNTs; its main novel feature is perhaps the proposition of chirality-dependent concepts of effective thickness and effective radius , whose evaluation is achieved in terms of nanoscopic information; moreover, as shown in the companion paper ( Favata and Podio-Guidugli, 2012b ), it can be generalized to fit SWCNTs of whatever chirality. • A shell theory of single-walled carbon nanotubes is proposed. • A notion of chirality-dependent thickness and radius is introduced. • Constitutive parameters are evaluated in terms of chirality.

Interaction of cisplatin and analogue Pt(en)Cl2 with the copper metallo-chaperone Atox1

The human metallo-chaperone protein Atox1 features a high affinity Cu(I) binding site Cys(12)GlyGlyCys(15) (KD = 10(-17.4) M at pH 7.0) and delivers copper to the trans-Golgi network (TGN). Atox1 may participate in the metabolism of the drug cis-Pt(NH3)2Cl2 (cisplatin), either as a component of its delivery to the nucleus or of its loss via transport to the TGN and beyond. The species of stoichiometry [Pt(NH3)2(Atox1)] was the sole adduct of stoichiometry Pt : Atox1 = 1 : 1 detected by mass spectrometry under non-denaturing conditions from solutions containing cisplatin and apo-Atox1. The ions [Atox1 + Pt(NH3)2(2+) + (z - 2)H(+)](z+) (z = 3 to 7) were observed and correspond to different protonation states of the 1 : 1 adduct. Adducts of stoichiometry Pt : Atox1 = 2 : 1 were also detected but 1 : 2 adducts were not detected. The related complex Pt(en)Cl2 (en = 1,2-diaminoethane) behaved similarly. Tandem mass spectrometry experiments using top-down and bottom-up sequencing techniques were carried out, respectively, on the intact platinated protein and on platinated peptides formed from proteolysis by trypsin. A new software programme (PolyCut) designed to analyse the complex high-resolution tandem mass spectra of fragment ions derived from proteins containing transition metal ions was applied to establish the binding site(s) of the platinum atom(s). The analysis, based on the entire isotope patterns, is consistent with the cysteine residues in the Cu(I)-binding sequence Cys(12)GlyGlyCys(15) being the primary coordination site.

Dissecting the size evolution of elliptical galaxies since z∼ 1: puffing-up versus minor-merging scenarios

We have explored the buildup of the local mass-size relation of elliptical galaxies using two visually classified samples. At low redshift we compiled a subsample of 2,656 elliptical galaxies from SDSS, whereas at higher redshift (up to z~1) we extracted a sample of 228 object from the HST/ACS images of the GOODS. All the galaxies in our study have spectroscopic data, allowing us to determine the age and mass of the stellar component. Using the fossil record information contained in the stellar populations of our local sample, we do not find any evidence for an age segregation at a given stellar mass depending on the size of the galaxies. At a fixed dynamical mass there is only a <9% size difference in the two extreme age quartiles of our sample. Consequently, the local evidence does not support a scenario whereby the present-day mass-size relation has been progressively established via a bottom-up sequence, where older galaxies occupy the lower part this relation, remaining in place since their formation. We find a trend in size that is insensitive to the age of the stellar populations, at least since z~1. This result supports the idea that the stellar mass-size relation is formed at z~1, with all galaxies populating a region which roughly corresponds to 1/2 of the present size distribution. The fact that the evolution in size is independent of stellar age, together with the absence of an increase in the scatter of the relationship with redshift does not support the puffing up mechanism. The observational evidence, however, can not reject at this stage the minor merging hypothesis. We have made an estimation of the number of minor merger events necessary to bring the high-z galaxies into the local relation compatible with the observed size evolution. Since z=0.8, if the merger mass ratio is 1:3 we estimate ~3+-1 minor mergers and if the ratio is 1:10 we obtain ~8+-2 events.

Utility of mass spectrometry for proteome ana lysis: part I. Conceptual and experimental approaches

This article is the first in a series of reviews intended as a tutorial providing the inexperienced, as well as the experienced, reader with an overview of principles of peptide and protein fragmentation in mass spectrometers for protein identification, surveying of the different types of instrument configurations and their combinations for protein identification. The first mass spectrometer was developed in 1899, but it took almost a century for the instrument to become a routine analytical method in proteomic research when fast atom bombardment ionization was developed, followed shortly by soft desorption/ionization methods, such as MALDI and electrospray ionization, to volatize biomolecules with masses of tens of kiloDaltons into the gas phase under vacuum pressure without destroying them. Thereafter, other soft ionization techniques that offered ambient conditions were also introduced, such as atmospheric pressure MALDI, direct analysis in real time, atmospheric-pressure solid analysis probe and hybrid ionization, sources of MALDI and electrospray ionization (e.g., two-step fused droplet electrospray ionization, laser desorption atmospheric-pressure chemical ionization, electrosonic spray ionization, desorption electrospray ionization, and electrospray-assisted laser desorption/ionization). The five basic types of mass analyzers currently used in proteomic research are the quadrupole, ion trap, orbitrap, Fourier transform ion cyclotron resonance and TOF instruments, which differ in how they determine the mass-to-charge ratios of the peptides. They have very different design and performance characteristics. These analyzers can be stand alone or, in some cases, put together in tandem or in conjunction with ion mobility mass spectrometry to take advantage of the strengths of each. Several singly or multiply charged fragment ion types, such as b, y, a, c, z, v, y and immonium ions are produced in the gas phase of the spectrometer. In the bottom-up sequencing approach for protein identification in a shotgun proteomic experiment, proteolytic digestion of proteins is accomplished by cleavage of the different bonds along the peptide backbone and/or side chain through a charge-directed transfer to the vicinity of the cleavage side. These various mass spectrometers and the types of ions produced have become important analytical tools for studying and analyzing proteins, peptides and amino acids.

Structural Analysis of Bikunin Glycosaminoglycan

The structure of an intact glycosaminoglycan (GAG) chain of the bikunin proteoglycan (PG) was analyzed using a combined top-down and bottom-up sequencing strategy. PGs are proteins with one or more linear, high-molecular weight, sulfated GAG polysaccharides O-linked to serine or threonine residues. GAGs are often responsible for the biological functions of PGs, and subtle variations in the GAG structure have pronounced physiological effects. Bikunin is a serine protease inhibitor found in human amniotic fluid, plasma, and urine. Bikunin is posttranslationally modified with a chondroitin sulfate (CS) chain, O-linked to a serine residue of the core protein. Recent studies have shown that the CS chain of bikunin plays an important role in the physiological and pathological functions of this PG. While no PG or GAG has yet been sequenced, bikunin, the least complex PG, offers a compelling target. Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FTICR-MS) permitted the identification of several major components in the GAG mixture having molecular masses in a range of 5505-7102 Da. This is the first report of a mass spectrum of an intact GAG component of a PG. FTICR-MS analysis of a size-uniform fraction of bikunin GAG mixture obtained by preparative polyacrylamide gel electrophoresis, allowed the determination of chain length and number of sulfo groups in the intact GAGs.

Proteases in Doping Control Analysis

Urine manipulation in sports drug testing has become a serious problem for doping control laboratories, and recent scandals in elite endurance sports have revealed the problem of urine manipulation presumably using proteases, which will impede the detection of drugs such as erythropoietin (EPO) or other peptide hormones. Using commonly accepted analytical strategies, a protocol was developed enabling the determination of elevated protease activities in doping control specimens followed by the visualization of protein degradation and identification of proteases such as chymotrypsin, trypsin and papain. Therefore, protease detection kits based on fluorescein isothiocyanate-labeled casein were employed, and protease concentrations greater than 15 microg/mL of urine entailed subsequent 1-dimensional gel electrophoretic visualization of urinary proteins. The presence of 20 microg of proteases per mL of urine caused a complete degradation of proteins usually observed in urinary matrices ("trace of burning"), while respective proteases were still detected in spiked urine samples after 10 days of storage at + 4 and - 20 degrees C. Identification of target proteases at respective molecular weights was accomplished using bottom-up sequencing approaches based on in-gel digestion of separated enzymes followed by capillary liquid chromatography--Orbitrap tandem mass spectrometry.

Kinematics of podokinetic after-rotation: Similarities to voluntary turning and potential clinical implications

We examined the kinematics of voluntary turning in place at three different speeds and of inadvertent turning in place during attempts to step in place following stepping on a rotating disc (podokinetic after-rotation, PKAR). We hypothesized that voluntary turning in place, like online turning during walking, would be characterized by a top-down sequence of yaw rotations in the direction of the turn, i.e. the head would rotate first, followed by the trunk and then the foot. We also hypothesized that in place PKAR would be characterized by a bottom-up sequence of yaw rotations, i.e. the foot would rotate first, followed by the trunk and the head. The alternative possibility was that PKAR, like voluntary turning, would be initiated by the head and trunk and the foot would rotate last. As expected, voluntary turning in place was characterized by a top-down sequence similar to that noted previously during online turning in the midst of walking. Turning velocity did not alter the sequence of rotations in voluntary turning. In place PKAR was also characterized by a top-down sequence, indicating that PKAR may access the same neural circuits employed during voluntary turning. These data suggest that the rotating treadmill may be a useful training tool for addressing difficulties with turning that are experienced by individuals with Parkinson disease (PD).

Mass spectrometry of peptides and proteins

This tutorial article introduces mass spectrometry (MS) for peptide fragmentation and protein identification. The current approaches being used for protein identification include top-down and bottom-up sequencing. Top-down sequencing, a relatively new approach that involves fragmenting intact proteins directly, is briefly introduced. Bottom-up sequencing, a traditional approach that fragments peptides in the gas phase after protein digestion, is discussed in more detail. The most widely used ion activation and dissociation process, gas-phase collision-activated dissociation (CAD), is discussed from a practical point of view. Infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) are introduced as two alternative dissociation methods. For spectral interpretation, the common fragment ion types in peptide fragmentation and their structures are introduced; the influence of instrumental methods on the fragmentation pathways and final spectra are discussed. A discussion is also provided on the complications in sample preparation for MS analysis. The final section of this article provides a brief review of recent research efforts on different algorithmic approaches being developed to improve protein identification searches.