Downstream Mass Research Articles

Optimized workflow of EV enrichment from human plasma samples for downstream mass spectrometry analysis

To improve the prognosis of bladder and prostate cancer, highly specific and sensitive biomarkers are needed for early detection, prognosis prediction, and therapeutic stratification. Extracellular vesicles (EV) from plasma could fill this gap due to their potential to serve as cancer biomarkers. However, the enrichment of EV is a major challenge, because the highly abundant plasma proteins are interfering with analytical downstream applications like mass spectrometry (MS). Therefore, the purity requirements of the EV samples must be carefully considered when selecting or developing a suitable EV enrichment method. The aim of this study was to compare a self-designed EV enrichment method based on density cushion centrifugation (DCC) combined with size exclusion chromatography (SEC) and concentration (method 1) with the exoRNeasy midi kit from Qiagen (method 2) and with unprocessed plasma. Furthermore, the single steps of method 1 were evaluated for their effectiveness to enrich EV from plasma. The results showed that the EV samples enriched with method 1 contained the highest levels of EV and exosome markers with simultaneously low levels of highly abundant plasma proteins. In summary, the combination of DCC, SEC and concentration proved to be a promising approach to discover EV-based biomarkers from plasma of cancer patients.

A highly stable, pressure-driven, flow control system based on Coriolis mass flow sensors for organs-on-chips

Stable delivery of liquids to microfluidic systems is essential for their reproducible functioning, especially when supplying flows to organs-on-chips – delicate living models that recreate human physiology on the microscale and thus can be used to reduce the need for animal testing. Most flow control systems are unable to sustain a robust and stable flow in longer experiments (>1 week), particularly those based on the ubiquitous syringe pump. Though easy to use, syringe pumps have no mechanism for actually measuring flow, let alone flow regulation with sensor feedback. We have developed a liquid delivery system based on the generation of flow by applying a constant air pressure to liquids in sealed containers. A flow of liquid is monitored by accurate measurement of mass flows (mg/min) using downstream Coriolis-based mass flow sensors. Measured mass flows provide fast feedback to integrated valves, with valves opening or closing slightly to increase or decrease solution flows to the organs-on-chips as required. This mass flow sensing principle is not affected by changes in the density, temperature, and viscosity of the liquids being displaced. This is in contrast to systems that use volumetric flow sensors, which require recalibration when these parameters change. The rationale behind using this principle for organs-on-chips, is that the stability provided by this flow control system allows for more control over growth of these mini-organs. We demonstrate the functionality of this system with three examples: 1) Fast stabilization (within seconds) under changing physical conditions; 2) Short-term stability (minutes to hours) of delivered flows in a microreactor with interconnected inlets; and 3) Long-term stability (>1 week) of cell medium flows to a living organ-on-a-chip. Two categories of organs-on-chips (OOCs) can be distinguished: 1) solid OOC are designed for three-dimensional cell or tissue constructs that interact with each other and their surroundings, and 2) barrier-type OOC contain a selective cellular barrier between two compartments as do many barriers in the body. The latter of these two types is the most challenging to culture and maintain as they are very sensitive to variations in flow and pressure surges. The flow control system presented in this work provides a great improvement compared to the use of syringe pumps and volumetric flow sensors in OOC studies. The novelty of this work lies in the long-term stability use of this system for organs-on-chips, maintaining stability for short to very long periods of time without compromising the barrier function of the organ-on-chip by pressure surges, bacterial contamination, or other undesired effects from the flow delivery system.

Open Port Interface for Coupling Capillary Electrophoresis and Mass Spectrometry: Performance Evaluation for Capillary Isoelectric Focusing.

Capillary electrophoresis (CE) combined with mass spectrometry (MS) is a powerful analytical technique that utilizes the resolving power of CE and the mass-detection capabilities of MS. In many cases, CE is coupled to MS via a sheath-flow interface (SFI). This interface has a simple design and can be easily constructed; however, it often suffers from issues such as MS signal suppression, interference of MS and CE electrical circuits, and the inability to set an optical point of detection close to the capillary end due to the specific design of the coupling union. In this paper, we describe a novel coupling of CE and MS based upon the open port interface (OPI). The OPI differs from classical sheath flow interfaces by operating at flow rates at least 1 order of magnitude higher. In addition to the flow rate difference, the OPI provides more efficient mixing of the capillary eluates with the transport fluid and thus minimizes MS signal suppression. In this work, we compared the performance of OPI and SFI in a series of capillary isoelectric focusing (cIEF) experiments with 5 pI markers, carbonic anhydrase II and NIST antibody. The evaluation criteria for the comparison of the OPI and SFI were analytical sensitivity, reproducibility, and pI marker linearity. Given the extent of sample dilution in the OPI, we also compared the peak resolution determined using an upstream UV detector to those determined by the downstream mass spectrometer. The results suggested that the OPI configuration reduced signal suppression, with no adverse effect on peak resolution. In addition, the OPI provided better decoupling of the CE and MS potentials as well as reduced signal dependence upon the sheath liquid composition. While these results are preliminary, they suggest that the OPI is a viable approach for CE-MS coupling.

Flame Surface Density and Artificially Thickened Flame Combustion Models Applied to a Turbulent Partially-Premixed Flame

The Sydney Flame provides an excellent framework for systematic analysis of premixed, partially premixed and non-premixed methane/air combustion. It represents (depending on the axial position from the fuel inlet) non-premixed, partially premixed and fully premixed combustion. We simulate the test case FJ200-5GP-Lr75-57, which represents the partially premixed mode. Large-eddy simulations (LES) are conducted using a flame surface density combustion model (FSD) using one-step chemistry and an artificially thickened flame model (ATF) with a global two-step and an analytically reduced multi-step chemistry reaction mechanism. Unity Lewis numbers are assumed (Le=1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$Le=1$$\\end{document}) at first. The FSD and ATF models are compared using the same computational mesh, numerical scheme and boundary conditions. Both models will be analysed regarding their accuracy and computational efficiency in the regime of partially premixed combustion. A comparison of the results points out the strengths and weaknesses of the FSD models. The FSD model with inclusion of flame stretch effects yields good agreement with mean experimental temperature, CO2 and H2O mass fraction distributions in contrast to the ATF model using the global two-step mechanism, which overestimates downstream temperature, CO2 and H2O mass fractions. The FSD model performs well in all regions, which are dominated first by premixed, then partially premixed and finally non-premixed combustion along the flame. The ATF multi-step chemistry shows good results only in the premixed mode region, while mean temperature, CO2 and mass fractions are overestimated in the non-premixed mode regions at higher mixture fraction values. Including differential diffusion into the transport equations improved the ATF model results in comparison with experiments. A mesh study revealed, that the ATF model performs only after mesh refinement. In particular results are improved in the non-premixed combustion region. In contrast, the FSD model is less resolution sensitive and performs very well for both meshes. An evaluation of the Wasserstein metric provides a quantitative assessment of different ATF model setups on simulation accuracy. Finally, computational times for all simulation setups are compared.

Spatially Resolving Heterogeneous Chemistry on SOFC Electrodes with Operando Optical Methods

Carbon fouling (or coking) is one of the primary degradation mechanisms that leads to performance loss and eventual failure in high temperature, solid oxide electrochemical cells (SOCs). Accumulated carbon blocks electrocatalytic sites at three phase boundaries, impedes transport through porous electrode structures and can react with common electrode materials such as Ni to form carbides that disintegrate in a process described as metal dusting. Operando Raman studies examining carbon remediation through gasification showed surprising results. (J. Phys. Chem. C, 119 (2015) 7637.) When comparing carbon removal from Ni-YSZ cermet anodes at 750˚C by gas phase H2O, O2, and CO2, experiments measuring intensity of the carbon ‘G’ band, Raman studies showed that H2O was most effective, removing all observable carbon in a matter of seconds. Similar amounts of O2 required approximately one minute to remove detectable carbon, and CO2 required even longer and never removed all of the observable carbon. These results were supported by changes in the SOC open circuit potential. These Raman measurements, however, sampled only a single location on the anode. Work described in this presentation shows the anode surface chemistry to be considerably more heterogeneous than might be inferred from the Raman data. A combination of near-IR thermal imaging (NIRTI), Fourier transform IR emission spectroscopy (FTIRES), voltammetry and downstream mass spectrometry (MS) exhaust analysis have begun to spatially resolve processes associated with carbon gasification from coked Ni-cermet anodes at 800˚C, and show that carbon gasification proceeds through a sequence of steps involving both surface and gas phase reactions.

Isolation of PD-L1 Extracellular Vesicle Subpopulations Using DNA Computation Mediated Microfluidic Tandem Separation.

Accurate isolation of targeted extracellular vesicle (EV) is challenging due to the antigenic heterogeneity of EV subpopulations which are from different cell origins. Most EV subpopulations lack a single marker whose expression cleanly distinguishes them from mixed populations of closely related EVs. Here, a modular platform capable of taking multiple binding events as input, performing logic computations, and producing two independent outputs for tandem microchips for EV subpopulation isolation, is developed. Taking advantages of the excellent selectivity of dual-aptamer recognition and the sensitivity of tandem microchips, this method achieves, for the first time, sequential isolation of tumor PD-L1 EVs and non-tumor PD-L1 EVs. As a result, the developed platform can not only effectively distinguish cancer patients from healthy donors but also provides new clues for assessing immune heterogeneity. Moreover, the captured EVs can be released through a DNA hydrolysis reaction with high efficiency, which is compatible with downstream mass spectrometry for EV proteome profiling. Overall, this strategy is expected to isolate different EV subpopulations, translate EVs into reliable clinical biomarkers, and accurately investigate the biological functions of different EV subsets.

Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma

Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry, requiring additional purification and buffer-exchange steps. This study compares two well-established detergent-based methods for protein extraction (in-solution sodium deoxycholate (SDC); suspension trapping (S-Trap)) with the recently developed sample preparation by easy extraction and digestion (SPEED) method, which uses strong acid for denaturation. We compared the quantitative performance of each method using label-free mass spectrometry in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED quantified the most unique proteins (SPEED 1250; S-Trap 1202; SDC 1197). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 150; SDC 148; SPEED 137). Protein quantifications were reproducible across biological replicates in both tissue (R2 = 0.85-0.90) and plasma (SPEED R2 = 0.84; SDC R2 = 0.76, S-Trap R2 = 0.65). Our data suggest SPEED as the optimal method for proteomic preparation in kidney tissue and S-Trap or SPEED as the optimal method for plasma, depending on whether a higher number of protein quantifications or greater reproducibility is desired.

Dissipating Culvert End Design for Erosion Control Using CFD Platform FLOW-3D Numerical Simulation Modeling

Downstream erosion and scouring caused by high-velocity flow issuing from culvert ends are one of the main problems faced by hydraulic engineers. The main objective of this paper was to develop a dissipating culvert end design that can reduce the risk of downstream erosion and scour caused by high-velocity flow issuing from typical culvert ends. For this purpose, the computational fluid dynamics (CFD) platform FLOW-3D version 11.1.0 code was calibrated and validated based on the experimental runs [coefficient of determination R2>0.90 and root mean square error (RMSE)<1.9 cm]. Two alternative dissipating culvert end designs (ALT 1 and ALT 2) were then developed using the code, and their potential in mitigation of downstream erosion and scouring was analyzed. The issuing flow velocity and kinetic energy for each were measured and compared with typical culvert end (control) flow. According to the results, mass averaged fluid mean kinetic energy in the control flow was recorded at 1.37 j/kg2 and was measured at 0.83 and 0.73 j/kg2 in ALT 1 and ALT 2 flows, respectively. Accordingly, the removal of downstream sandbox mass under control flow was approximately 11.1% and 4.2% higher compared with ALT 1 and ALT 2 flows, respectively. FLOW-3D code can be used to predict culvert end flow and downstream erosion and to design potential dissipating culvert ends that can reduce downstream erosion.

RNA and Protein Interactomes of an RNA-Binding Protein Tagged with FLAG Epitopes Using Combinatory Approaches of Genome Engineering and Stable Transfection.

To study the function of RNA-binding proteins (RBPs), an overexpression or knockout approach is generally used. However, as many RBPs are essential to cellular functions, the complete knockout of these proteins may be lethal to the cell. Overexpression of RBPs, on the other hand, may create an altered transcriptome and aberrant phenotypes that can mask their physiological function. Additionally, biochemical characterization of RBP often requires highly specific antibodies for efficient immunoprecipitation for downstream mass spectrometry or RNA footprinting profiling. To overcome these hurdles, we have developed a strategy to generate cellular systems either using a CRISPR-Cas9-mediated epitope tag knock-in approach or a two-step workflow to first stably express an exogenous Flag-tagged RBP and subsequently knockout the endogenous RBP using CRISPR-Cas9 gene editing. The generation of these cell lines will be beneficial for downstream RNA footprinting studies and mass spectrometry-mediated interactome studies.

Metabolite Extraction from RPE Cells and Retinas Related to Retinitis Pigmentosa.

The application of metabolomics in ophthalmology helps to identify new biomarkers and elucidate disease mechanisms in different eye diseases, as well as aiding in the development of potential treatment options. Extracting metabolites successfully is essential for potential further analysis using mass spectrometry. In this chapter, we describe how to extract metabolites from a variety of sources including (1) cells on a dish, (2) cell culture medium, and (3) tissues in vivo with and without stable isotope tracers. Samples prepared using this protocol are suitable for a range of downstream mass spectrometry analyses and are stable in solvent for weeks at -80°C.

DIGE-Based Biomarker Discovery in Blood Cancers.

Cancer of blood or bone marrow-derived cells dysregulates normal hematopoiesis and accounts for over 6% of all cancer cases annually. Proteomic analyses of blood cancers have improved our understanding of disease mechanisms and identified numerous proteins of clinical relevance. For many years, gel-based proteomic studies have aided in the discovery of novel diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, in various diseases, including blood cancer. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) facilitates comparative proteomic research to identify differential protein expression in a simple and reproducible manner. The versatility of 2D-DIGE as a quantitative proteomic technique has provided insight into various aspects of blood cancer pathology, including disease development, prognostic subtypes, and drug resistance. The ability to couple 2D-DIGE with additional downstream mass spectrometry-based techniques yields comprehensive workflows capable of identifying proteins of biological and clinical significance. The application of 2D-DIGE in blood cancer research has significantly contributed to the increasingly important initiative of precision medicine. This chapter will focus on the influential role of 2D-DIGE as a tool in blood cancer research.

Estimation of Steroid Hormones in Biological Samples Using Micro Extraction and Advanced Chromatography Techniques

Steroid profiling plays an important role in the clinical setting and also in the diagnosis of various physiological disorders. Steroids in the research laboratory and patient care has been routinely measured by immunoassay. Limitations of immunoassay in quantifying steroids have been well documented and the advent of advanced mass spectrometry is offering a viable alternative to measure multiple steroids in single reaction. Analytes ranging from the steroid and their metabolites that are present in the body carry out various important functions and are relevant to maintaining homeostasis. Blood plasma and urine samples are the clinical material for such analysis, however, due to the difference in polarity of the target steroid, a major challenge exists as to how faithfully analyze different steroids in the given sample type. These steroids are usually present at very low concentrations in the body and are present in several forms leading to increased complexity. Achieving an excellent chromatographic separation for the analysis of steroids requires an effective sample preparation and analysis procedure. Hence various methods are developed to analyze the presence of steroids and to know their exact concentration in the body. This review covers various analytical methodologies such as chromatographic procedures and downstream mass spectrometry for the identification and measurement of the levels of steroid hormones. At the same time, this review also covers the most recent developments that have taken place in the recent past to cover the enhanced understanding of steroid hormone analysis.

Impact of the downstream myocardial mass on values of coronary microvascular resistance.

The assessment of hyperemic microvascular resistance (HMR) may be dependent on the assessment location in the coronary artery and the amount of partial myocardial mass (PMM) distal to the assessment locations. The aim of this study was to investigate the differences in HMR values between the distal and proximal sites in the same coronary arteries as well as the relationship between HMR and PMM. Twenty-nine vessels from 26 patients who had undergone intracoronary physiological assessments including Doppler flow velocity at the distal third part and the proximal third part in the same vessels were assessed. The mean values of HMR and PMM at the distal sites were 2.08 ± 0.75 mmHg/cm/sec and 22.2 ± 10.4g, respectively. At the proximal sites, the values of HMR and PMM were 1.19 ± 0.33 mmHg/cm/sec and 59.9 ± 18.3g, respectively. All HMR values at the distal sites were significantly higher than those at the proximal sites (p &lt; 0.001). Smaller PMM at the distal sites was significantly associated with higher HMR (r=-0.544, p=0.002) and was the strongest factor affecting the HMR values (p=0.009), while this relationship was not observed at the proximal sites (r=-0.262, p=0.17). The impact of PMM on HMR was diminished at assessment locations where PMM was greater than 35 g. In conclusion, a small amount of downstream myocardial mass could be related to high HMR values. The assessment location around the proximal coronary artery with over 35 g of myocardium would be appropriate to assess HMR because it minimizes the influence of the assessment location.

Ionization and current sheet formation in inductive pulsed plasma thrusters

Current sheet formation in inductive pulsed plasma thrusters (IPPTs) is investigated theoretically to determine how non-equilibrium ionization processes influence plasma impermeability to inductive electromagnetic fields and downstream propellant mass. Plasma impermeability to both electromagnetic fields and downstream mass is a prerequisite for efficient IPPT operation. A lumped-element circuit model of an IPPT plasma is modified to include propellant ionization and the electron energy balance under non-equilibrium conditions, neutral gas entrainment via charge exchange collisions, and electromagnetic coupling to a finite skin depth plasma. It is found that current sheets impermeable to both the accelerating fields and downstream mass—presumed to exist in all prior circuit modeling efforts—form only under specific conditions. The dynamics of electron heating during the early portion of the inductive current cycle are identified as the dominant contributors to current sheet formation. A new dimensionless scaling parameter is derived to characterize electron heating relative to inelastic ionization losses, from which it is found that impermeable current sheet formation requires Ohmic heating in the early formation phase to offset ionization losses associated with the entire propellant mass bit. This finding provides a physical explanation and generalization of the semi-empirical requirement on coil current rise rate that is commonly used in the early design phase of IPPTs to ensure current sheet formation.

The Throttle Effect – Blower Fan Versus Exhaust Fan

Abstract One of the risks connected to fires underground is the throttle effect which may cause unforeseen smoke spread. This paper investigates the throttle effect for a blower fan and an exhaust fan case in a mine drift. The aim of the paper is to perform a parametric study on the throttle effect, varying influencing parameters such as the heat release rate and fan flow velocity. Data from fire experiments in a model-scale mine drift and results from CFD simulations were used during the study. It was found that the differences between the two fan cases were significant both in magnitude and occasionally in direction. For the base cases the throttle effect as well as the backlayering were more severe in the exhaust fan case. When increasing the heat release rate to 116 kW an increasing backlayering resulted, but the throttle effect was found to increase for the exhaust fan case and decrease for the blower fan case. The throttle effect decreased in the blower fan case as the gas density decrease levelled off, but the flow velocity increased even further, causing an increase in the downstream mass flow rate. This finding was confirmed by similar experimental results in model-scale mine drifts. The resulting mass flow rate induced by the fire plume changes was found to be higher than the externally imposed increase of the fan flow velocity. When increasing the distance between the fire and the exhaust fan, the backlayering increased and the throttle effect decreased.

An R-based reproducible and user-friendly preprocessing pipeline for CyTOF data

Mass cytometry (CyTOF) has become a method of choice for in-depth characterization of tissue heterogeneity in health and disease, and is currently implemented in multiple clinical trials, where higher quality standards must be met. Currently, preprocessing of raw files is commonly performed in independent standalone tools, which makes it difficult to reproduce. Here, we present an R pipeline based on an updated version of CATALYST that covers all preprocessing steps required for downstream mass cytometry analysis in a fully reproducible way. This new version of CATALYST is based on Bioconductor’s SingleCellExperiment class and fully unit tested. The R-based pipeline includes file concatenation, bead-based normalization, single-cell deconvolution, spillover compensation and live cell gating after debris and doublet removal. Importantly, this pipeline also includes different quality checks to assess machine sensitivity and staining performance while allowing also for batch correction. This pipeline is based on open source R packages and can be easily be adapted to different study designs. It therefore has the potential to significantly facilitate the work of CyTOF users while increasing the quality and reproducibility of data generated with this technology.

Rapid and In-Depth Coverage of the (Phospho-)Proteome With Deep Libraries and Optimal Window Design for dia-PASEF

Data-independent acquisition (DIA) methods have become increasingly attractive in mass spectrometry–based proteomics because they enable high data completeness and a wide dynamic range. Recently, we combined DIA with parallel accumulation–serial fragmentation (dia-PASEF) on a Bruker trapped ion mobility (IM) separated quadrupole time-of-flight mass spectrometer. This requires alignment of the IM separation with the downstream mass selective quadrupole, leading to a more complex scheme for dia-PASEF window placement compared with DIA. To achieve high data completeness and deep proteome coverage, here we employ variable isolation windows that are placed optimally depending on precursor density in the m/z and IM plane. This is implemented in the freely available py_diAID (Python package for DIA with an automated isolation design) package. In combination with in-depth project-specific proteomics libraries and the Evosep liquid chromatography system, we reproducibly identified over 7700 proteins in a human cancer cell line in 44 min with quadruplicate single-shot injections at high sensitivity. Even at a throughput of 100 samples per day (11 min liquid chromatography gradients), we consistently quantified more than 6000 proteins in mammalian cell lysates by injecting four replicates. We found that optimal dia-PASEF window placement facilitates in-depth phosphoproteomics with very high sensitivity, quantifying more than 35,000 phosphosites in a human cancer cell line stimulated with an epidermal growth factor in triplicate 21 min runs. This covers a substantial part of the regulated phosphoproteome with high sensitivity, opening up for extensive systems-biological studies.

Enhanced semi-volatile DNAPL accessibility at sub-boiling temperature during electrical resistance heating in heterogeneous porous media

Successful remediation of semi-volatile contaminants using electrical resistance heating (ERH) coupled technologies requires a deep understanding of contaminant migration and accessibility, especially with stratigraphic heterogeneity and dense nonaqueous phase liquid (DNAPL) occurrence. Here, we chose nitrobenzene (NB) as a model contaminant of semi-volatile DNAPL and uniquely demonstrated that temperature variation during ERH could induce NB DNAPL migration out of the low permeability zone (LPZ) even below water boiling temperature. When heating the system using alternating current (AC) of 140 V to a temperature range of 50–79 °C, obvious DNAPL migration was visually observed. The upward migration of DNAPL would considerably increase the mass of accessible contaminant by other remediation measures. The downstream cumulative NB mass of 1092 mg in 140 V system raised 56-folds compared to that of 19 mg in the control experiment with only groundwater flow. This migration was mainly attributed to a complex natural convection caused by temperature gradient. Comparing with traditional AC heating, ERH powered by pulsed direct current (PDC-ERH) showed a higher and more uneven heating pattern, resulting in a stronger convection at the same voltage that enhanced the DNAPL migration out of LPZ. These results revealed the importance of natural convection in the ERH process, which could be further optimized to improve the energy efficiency of remediation.

Optimization of a detergent-based protocol for membrane proteins purification from mammalian cells

Membrane proteins constitute around 20–30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of “omics” techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers.

Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosis.

ABSTRACTStudies involving the pathogenic organism Mycobacterium tuberculosis routinely require advanced biosafety laboratory facilities, which might not be readily available in rural areas where tuberculosis burdens are high. Attempts to adapt heat inactivation techniques have led to inconsistent conclusions, and the risk of protein denaturation due to extensive heating is impractical for subsequent mass spectrometry (MS)-based protein analyses. In this study, 240 specimens with one or two loops of M. tuberculosis strain H37Rv biomass and specific inactivated solutions were proportionally assigned to six heat inactivation methods in a thermal block at 80°C and 95°C for 20, 30, and 90 min. Twenty untreated specimens served as a positive control, and bacterial growth was followed up for 12 weeks. Our results showed that 90 min of heat inactivation was necessary for samples with two loops of biomass. Further protein extraction and a matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS assay demonstrated adequate scores for bacterial identification (≥1.7), with the highest score achieved in the 80°C/90 min and 95°C/30 min treatment groups. A proteomics study also confidently identified 648 proteins with ∼93% to 96% consistent protein abundances following heating at 95°C for 20, 30, and 90 min. Heat inactivation at 95°C for 90 min yielded the most quantifiable proteins, and a functional analysis revealed proteins located in the ribosomal subunit. In summary, we proposed a heat inactivation method for the M. tuberculosis strain H37Rv and studied the preservation of protein components for subsequent bacterial identification and protein-related assays.IMPORTANCE Inactivation of Mycobacterium tuberculosis is an important step to guarantee biosafety for subsequent M. tuberculosis identification and related research, notably in areas of endemicity with minimal resources. However, certain biomolecules might be denatured or hydrolyzed because of the harsh inactivation process, and a standardized protocol is yet to be determined. We evaluated distinct heating conditions to report the inactivation efficiency and performed downstream mass spectrometry-based M. tuberculosis identification and proteomics study. The results are important and useful for both basic and clinical M. tuberculosis studies.