Improved Fragmentation Efficiency Research Articles

Small-Scale Serial Size Exclusion Chromatography (s3SEC) for High Sensitivity Top-Down Proteomics of Large Proteoforms.

Top-down-mass spectrometry (MS)-based proteomics has emerged as a premier technology to examine proteins at the proteoform level, enabling characterization of genetic mutations, alternative splicing, and post-translational modifications. However, significant challenges that remain in top-down proteomics include the analysis of large proteoforms and the sensitivity required to examine proteoforms from minimal amounts of sample. To address these challenges, we have developed a new method termed "small-scale serial Size Exclusion Chromatography" (s3SEC), which incorporates a small-scale protein extraction (1 mg of tissue) and serial SEC without postfractionation sample handling, coupled with online high sensitivity capillary reversed-phase liquid chromatography tandem MS (RPLC-MS/MS) for analysis of large proteoforms. The s3SEC-RPLC-MS/MS method significantly enhanced the sensitivity and reduced the proteome complexity across the fractions, enabling the detection of high MW proteoforms previously undetected in one-dimensional (1D)-RPLC analysis. Importantly, we observed a drastic improvement in the signal intensity of high MW proteoforms in early fractions when using the s3SEC-RPLC method. Moreover, we demonstrate that this s3SEC-RPLC-MS/MS method also allows the analysis of lower MW proteoforms in subsequent fractions without significant alteration in proteoform abundance and equivalent or improved fragmentation efficiency to that of the 1D-RPLC approach. Although this study focuses on the use of cardiac tissue, the s3SEC-RPLC-MS/MS method could be broadly applicable to other systems with limited sample inputs.

Does Moses technology improve the efficiency and outcomes of standard holmium laser lithotripsy? A systematic review and meta-analysis.

Retrograde ureteroscopy with holmium laser lithotripsy (HLL) is a standard treatment for urolithiasis. Moses technology has been shown to improve fragmentation efficiency in vitro; however, it is still unclear how it performs clinically compared to standard HLL. We performed a systematic review and meta-analysis evaluating the differences in efficiency and outcomes between Moses mode and standard HLL. We searched the MEDLINE, EMBASE, and CENTRAL databases for randomized clinical trials and cohort studies comparing Moses mode and standard HLL in adults with urolithiasis. Outcomes of interest included operative (operation, fragmentation, and lasing times; total energy used; and ablation speed) and perioperative parameters (stone-free rate and overall complication rate). The search identified six studies eligible for analysis. Compared to standard HLL, Moses was associated with significantly shorter average lasing time (mean difference [MD] -0.95, 95% confidence interval [CI] -1.22 to -0.69 minutes), faster stone ablation speed (MD 30.45, 95% CI 11.56-49.33 mm3/min), and higher energy used (MD 1.04, 95% CI 0.33-1.76 kJ). Moses and standard HLL were not significantly different in terms of operation (MD -9.89, 95% CI -25.14 to 5.37 minutes) and fragmentation times (MD -1.71, 95% CI -11.81 to 8.38 minutes), as well as stone-free (odds ratio [OR] 1.04, 95% CI 0.73-1.49) and overall complication rates (OR 0.68, 95% CI 0.39-1.17). While perioperative outcomes were equivalent between Moses and standard HLL, Moses was associated with faster lasing time and stone ablation speeds at the expense of higher energy usage.

V03-01 UNDERSTANDING NON-CONTACT LASER LITHOTRIPSY FOR DUSTING – THE POPCORN EFFECT: A VIDEO ANALYSIS

You have accessJournal of UrologyUrolithiasis & Endourology1 Apr 2018V03-01 UNDERSTANDING NON-CONTACT LASER LITHOTRIPSY FOR DUSTING – THE POPCORN EFFECT: A VIDEO ANALYSIS Ali Aldoukhi, William Roberts, Timothy Hall, and Khurshid Ghani Ali AldoukhiAli Aldoukhi More articles by this author , William RobertsWilliam Roberts More articles by this author , Timothy HallTimothy Hall More articles by this author , and Khurshid GhaniKhurshid Ghani More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.826AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The mechanism of stone breakdown and factors affecting fragmentation during non-contact holmium laser lithotripsy (the popcorn effect) at ureteroscopy (URS) are not fully understood. Using an in vitro model, we undertook high speed video analysis to better understand this mechanism and determine the effects of laser settings, calyceal size, flow rate, time, and heat generation on sub-millimeter fragmentation (i.e. dusting). METHODS BegoStones (15:3) of fixed size were pulverized in a test tube calyceal model of various sizes (11, 15, and 19 mm) using a 120 W holmium laser (P120, Lumenis). A 200 μm laser fiber (Flexiva 200, Boston Scientific) was introduced through the working channel of a URS (LithoVue, Boston Scientific) connected to a 3D positioning system. The fiber was placed 1-2 mm away from the stones. Irrigation was provided at a height of 100 cm. Four different settings were assessed (1.0J x 20-40Hz, 0.5J x 40-80Hz). Video analysis was undertaken using a Photron high speed camera (10,000 and 500 frames per second). Experiments to determine optimal laser setting were conducted for 4 minutes (with 30 second intervals) and subsequent experiments were done at 3 minutes utilizing the best setting - assessing impact of calyceal size, flow rate and time. Fragments were sieved and the primary outcome was % fragments <1 mm. Temperature was recorded at the end of 30 seconds of laser activation using a thermocouple. RESULTS Video analysis revealed that fragmentation occurred when the stones were in contact with the laser fiber and not when the stones collided. Stone movement occurred as a result of pressure waves generated from vapor bubble expansion and collapse. Superior submillimeter fragmentation was achieved using 0.5J x 80Hz. Short pulse width resulted in significantly greater fragmentation than long pulse. Smaller calyceal sizes led to improved motion and fragmentation. A linear increase in fragmentation over time was observed; however, efficiency decreased after 4 minutes. There was a minimal role of flow rate on outcomes. Temperature reached 50 °C after 30 seconds of continuous firing for 40 W settings, but only 45 °C when activated for 10 seconds. CONCLUSIONS The mechanism of fragmentation during popcorning is by stone to laser fiber interaction. Stone movement is necessary to improve fragmentation efficiency, and smaller calyceal model sizes led to better results. Superior submillimeter fragmentation was achieved using the 40 W setting (0.5 J x 80 Hz). However, 40 W settings generated more heat so the laser should be activated in short bursts to reduce the risk of thermal injury. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e321 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Ali Aldoukhi More articles by this author William Roberts More articles by this author Timothy Hall More articles by this author Khurshid Ghani More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A high resolution atmospheric pressure matrix-assisted laser desorption/ionization-quadrupole-orbitrap MS platform enables in situ analysis of biomolecules by multi-mode ionization and acquisition

Introduced in 2000, atmospheric pressure (AP)/matrix-assisted laser desorption/ionization (MALDI) has attracted substantial attention in the mass spectrometry community due to its ease of sample introduction and handling, interchangeability with ESI source and capability of analyzing volatile species. In this study, an AP/MALDI source with ultra-high spatial resolution was coupled to a Q Exactive HF orbitrap mass spectrometer for high resolution in situ analysis by MALDI, laserspray ionization (LSI) and matrix assisted ionization (MAI) without instrument modification. LSI and MAI generated multiply charged ions, which expanded the mass detection range and improved fragmentation efficiency. Full MS, targeted MS/MS, data dependent acquisition (DDA) and parallel reaction monitoring (PRM) acquisitions were performed on peptide and protein standards, tissue extracts and tissue sections for in depth characterization of various biomolecules. High resolution full MS and MS/MS images were obtained from crustacean and rat tissues with pixel size less than 30 μm. Overall, AP/MALDI-Q-Orbitrap is a fast scanning instrument that is capable of performing multiple types of ionization and multiple acquisition modes without instrument modification. This instrument platform provides an attractive alternative to other high resolution MALDI instruments.

Matrix-assisted ionization vacuum for protein detection, fragmentation and PTM analysis on a high resolution linear ion trap-orbitrap platform

Matrix-assisted ionization vacuum (MAIV) is a novel ionization technique that generates multiply charged ions in vacuum without the use of laser ablation or high voltage. MAIV can be achieved in intermediate-vacuum and high-vacuum matrix-assisted laser desorption/ionization (MALDI) sources and electrospray ionization (ESI) sources without instrument modification. Herein, we adapt MAIV onto the MALDI-LTQ-Orbitrap XL platform for biomolecule analysis. As an attractive alternative to MALDI for in solution and in situ analysis of biomolecules, MAIV coupling to high resolution and accurate mass (HRAM) MS instrument has successfully expanded the mass detection range and improved the fragmentation efficiency due to the generation of multiply charged ions. Additionally, the softness of MAIV enables potential application in labile post-translational modification (PTM) analysis. In this study, proteins as large as 18.7 kDa were detected with up to 18 charges; intact peptides with labile PTM were well preserved during the ionization process and characterized MS/MS; peptides and proteins in complex tissue samples were detected and identified both in liquid extracts and in situ. Moreover, we demonstrated that this method facilitates MS/MS analysis with improved fragmentation efficiency compared to MALDI-MS/MS.

Feasibility study on a short-pulsed IR wavelength for effective calculus fragmentation

Laser-induced lithotripsy has been used for a minimally-invasive surgery to treat kidney-stone disease associated with urinary obstruction. A short-pulsed Tm:YAG laser (λ = 2.01 µm) was developed to improve fragmentation efficiency and was evaluated with a Ho:YAG laser (λ = 2.12 μm) as to its ablation feature and mass removal rate. Application of a train of sub-microsecond pulses with a lower energy at a frequency of 500 Hz created multiple events of cavitation that accompanied strong acoustic transients. During Tm:YAG irradiation, both high light absorption and secondary photomechanical impacts readily fragmented the calculus into small pieces (< 3 mm) and removed them 130 times faster than photothermal Ho:YAG lithotripsy. The proposed short-pulsed Tm:YAG approach may be an effective lithotripter for treating calculus disease.

Numerical modelling of heterogeneous rock breakage behaviour based on texture images

The main objective of this study is to understand rock breakage behaviours which may provide a means to improve fragmentation efficiency. In this study, numerical modelling technology and image analysis are used to investigate the behaviour of actual rocks in the breakage process. A texture-based finite element method (FEM) modelling technique has been developed to present a realistic modelling method for characterizing heterogeneous rock breakage behaviour according to its actual microstructure using integrated microscopic observation, image analysis and numerical modelling.Understanding the role of texture and crack propagation can provide fundamental knowledge for predicting and improving energy-efficiency of comminution. A number of samples were analysed with the aim of providing key information on mineralogical and textural controls which primarily influence preferential liberation.The results in this study demonstrated that mineral distributions and minerals with lower tensile strength play a very important role in the breakage process.

N,N-Dimethyl Leucines as Novel Isobaric Tandem Mass Tags for Quantitative Proteomics and Peptidomics

Herein, we describe the development and application of a set of novel N,N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass (MS(2)) tagging reagents with high quantitation efficacy and greatly reduced cost for neuropeptide and protein analysis. DiLeu reagents serve as attractive alternatives for isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMTs) due to their synthetic simplicity, labeling efficiency, and improved fragmentation efficiency. DiLeu reagent resembles the general structure of a tandem mass tag in that it contains an amine reactive group (triazine ester) targeting the N-terminus and epsilon-amino group of the lysine side chain of a peptide, a balance group, and a reporter group. A mass shift of 145.1 Da is observed for each incorporated label. Intense a(1) reporter ions at m/z 115.1, 116.1, 117.1, and 118.1 are observed for all pooled samples upon MS(2). All labeling reagents are readily synthesized from commercially available chemicals with greatly reduced cost. Labels 117 and 118 can be synthesized in one step and labels 115 and 116 can be synthesized in two steps. Both DiLeu and iTRAQ reagents show comparable protein sequence coverage (approximately 43%) and quantitation accuracy (<15%) for tryptically digested protein samples. Furthermore, enhanced fragmentation of DiLeu labeling reagents offers greater confidence in protein identification and neuropeptide sequencing from complex neuroendocrine tissue extracts from a marine model organism, Callinectes sapidus.

In Vitro Evaluation of Nitinol Urological Retrieval Coil and Ureteral Occlusion Device: Retropulsion and Holmium Laser Fragmentation Efficiency

Retropulsion of ureteral stones during laser lithotripsy may result in difficult and incomplete stone fragmentation. The Stone Cone nitinol urological retrieval coil and the NTrap nitinol ureteral occlusion device have been introduced into clinical practice to possibly limit stone retropulsion and enhance the efficiency of holmium laser (Convergent Laser Technologies, Alameda, California) stone fragmentation. A total of 360 BegoStone Plus phantom stones (Bego USA, Smithfield, Rhode Island) of similar mass and weight were divided into 3 groups, including control, Stone Cone and NTrap. The groups were further subdivided according to fiber size (200 or 400 microm) and pulse width (350 or 700 microsec). These stones were placed in a horizontal pipette 12 mm in diameter, submerged in normal saline and disintegrated at laser settings of 1 J and 10 Hz continuously applied for 300 seconds. Retropulsion in cm and fragmentation efficiency with mass loss in mg were measured after treatment. The 2 devices were effective for preventing retropulsion. In the control group the mean +/- SD retropulsion distance using a 350-microsec pulse width with the 200 and 400 microm fibers was 18.4 +/- 5.9 and 14.1 +/- 4.6 cm, while it was 6.2 +/- 2.6 and 5.6 +/- 2.4, respectively, using the 700-microsec pulse width. There was a statistically significant higher loss of stone weight in the Stone Cone and NTrap experimental groups than in the control group (p <0.0001). However, there was no difference between the 2 experimental groups across all groups (p = 0.32). The Stone Cone and NTrap eliminated retropulsion and equally improved fragmentation efficiency. The maximum efficiency of fragmentation was seen using the 200 microm fiber at a 700-microsec pulse width.

The Electromechanical Impactor: The Results of Design Modifications

The first generation clinical electromechanical impactor was a 5F device that used a 3.0F electrohydraulic probe within a coiled spring with a blunt solder end cap. Clinical trials showed that this device fragmented 70% of the calculi but failed to fragment the harder stones. Applying the principle that impact kinetic energy is most dependent on velocity (KE = 1/2 MV2), 3 major design modifications were made to improve fragmentation efficiency: 1) the spring was changed from an extension to a compression type that captured and used more of each electrical spark, 2) the tip was changed from solder to lightweight titanium and 3) the tip shape was changed from blunt to conical. The result was a 41% increase in impact kinetic energy, a 40% increase in probe longevity and successful in vitro fragmentation of the harder calculi without compromise in tissue safety as determined by membrane perforation studies.

Acoustic studies of the role of immersion in plasma-mediated laser ablation

Several experiments were performed to understand the increased efficiency of microsecond-domain pulsed visible laser fragmentation of urinary and biliary stones produced by immersion in water. An optoacoustic sensor and a piezoelectric transducer were used to measure the effect of water immersion on the stress wave traveling through a stone during laser-induced ablation. The stress wave signal increased by a factor of 10 with immersion and was proportional to the square root of laser fluence. The temporal and spatial behavior of the stress wave and cavity (bubble) generated in the surrounding water were examined using pump-probe and microsecond-flash photographic techniques. The results suggest that improved fragmentation efficiency can be explained by confinement of a plasma by water.