SDS Buffer Research Articles

Biochemical Fractionation of Human α-Synuclein in a Drosophila Model of Synucleinopathies.

Synucleinopathies are a group of central nervous system pathologies that are characterized by the intracellular accumulation of misfolded and aggregated α-synuclein in proteinaceous depositions known as Lewy Bodies (LBs). The transition of α-synuclein from its physiological to pathological form has been associated with several post-translational modifications such as phosphorylation and an increasing degree of insolubility, which also correlate with disease progression in post-mortem specimens from human patients. Neuronal expression of α-synuclein in model organisms, including Drosophila melanogaster, has been a typical approach employed to study its physiological effects. Biochemical analysis of α-synuclein solubility via high-speed ultracentrifugation with buffers of increasing detergent strength offers a potent method for identification of α-synuclein biochemical properties and the associated pathology stage. Unfortunately, the development of a robust and reproducible method for the evaluation of human α-synuclein solubility isolated from Drosophila tissues has remained elusive. Here, we tested different detergents for their ability to solubilize human α-synuclein carrying the pathological mutation A53T from the brains of aged flies. We also assessed the effect of sonication on the solubility of human α-synuclein and optimized a protocol to discriminate the relative amounts of soluble/insoluble human α-synuclein from dopaminergic neurons of the Drosophila brain. Our data established that, using a 5% SDS buffer, the three-step protocol separates cytosolic soluble, detergent-soluble and insoluble proteins in three sequential fractions according to their chemical properties. This protocol shows that sonication breaks down α-synuclein insoluble complexes from the fly brain, making them soluble in the SDS buffer and thus enriching the detergent-soluble fraction of the protocol.

Comparison of two Genomic DNA Extraction Protocols from Single Dray Seed of Barley Crop

Modern plant breeding studies are largely based on plant genetic engineering programs. Extraction of DNA with high quality plays as a key factor in most of plant genetic Studies, therefore two different DNA extraction protocols based on CTAB buffer and SDS buffer were tested for the purpose of selecting the best DNA extraction protocol from dry seeds of nine variety of barely plant . Barley seeds were taken from Libyan seeds gene bank .After Barely samples were prepared DNA was extracted directly using CTAB and SDS solutions .The quality of extracted DNA was assessed by spectrophotometric measurements and gel electrophoresis system. The results of this study showed that purity of extracted DNA by CTAB method was clearly batter compared with SDS method. CTAB method seemed to be more effective for extracting DNA from barely dry seeds. High quality DNA obtained through use of CTAB method, while CTAB had overall better A260/A280 ratio (1.736-1.932). SDS method seemed to be not suitable for DNA extraction from dry seeds of barely crop.

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS.

The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.

Sonication-Assisted Method for Decellularization of Human Umbilical Artery for Small-Caliber Vascular Tissue Engineering.

Decellularized vascular grafts are useful for the construction of biological small-diameter tissue-engineered vascular grafts (≤6 mm). Traditional chemical decellularization requires a long treatment time, which may damage the structure and alter the mechanical properties. Decellularization using sonication is expected to solve this problem. The aim of this study was to develop an effective decellularization method using ultrasound followed by washing. Different power values of sonication at 40 kHz were tested for 2, 4, and 8 h followed by a washing procedure. The efficacy of sonication of decellularized human umbilical artery (sDHUA) was evaluated via DNA content, histological staining, mechanical properties, and biocompatibility. The sDHUAs were further implanted into rats for up to 90 days and magnetic resonance angiography (MRA) was performed for the implanted grafts. The results demonstrated that treatment of human umbilical artery (HUA) by sonication at ultrasonic power of 204 W for 4 h followed by washing for 24 h in 2% SDS buffer could eliminate more than 90% of cells and retain similar mechanical properties of the HUA. Recellularization was assessed by scanning electron microscopy (SEM), which indicated that sDHUA provided niches for human umbilical vein endothelial cells (HUVECs) to reside, indicating in vitro cytocompatibility. Further implantation tests also indicated the fitness of the sonication-treated HUA as a scaffold for small-caliber tissue engineering vascular grafts.

Online top-down mass spectrometric identification of CE(SDS)-separated antibody fragments by two-dimensional capillary electrophoresis

Size heterogeneity analysis by capillary sieving electrophoresis utilizing sodium dodecyl sulfate (CE(SDS)) with optical detection is a major method applied for release and stability testing of monoclonal antibodies (mAbs) in biopharmaceutical applications. Identification of mAb-fragments and impurities observed with CE(SDS) is of outstanding importance for the assessment of critical quality attributes and development of the analytical control system. Mass spectrometric (MS) detection is a powerful tool for protein identification and characterization. Unfortunately, CE(SDS) is incompatible with online MS-hyphenation due to strong ionization suppression of SDS and other separation buffer components.Here, we present a comprehensive platform for full characterization of individual CE(SDS)-separated peaks by CE(SDS)-capillary zone electrophoresis-top-down-MS. The peak of interest is transferred from the first to the second dimension via an 8-port valve to remove MS-incompatible components. Full characterization of mAb byproducts is performed by intact mass determination and fragmentation by electron transfer dissociation, higher-energy collisional dissociation, and ultraviolet photodissociation. This enables online determination of intact mass as well as sequence verification of individual CE(SDS)-separated peaks simultaneously. A more substantiated characterization of unknown CE(SDS) peaks by exact localization of modifications without prior digestion is facilitated. High sensitivity is demonstrated by successful mass and sequence verification of low abundant, unknown CE(SDS) peaks from two stressed mAb samples. Good fragmentation coverages are obtained by MS2, enabling unequivocal identification of these mAb-fragments. Also, the differentiation of reduced/non-reduced intra-protein disulfide bonds is demonstrated. In summary, a reliable and unambiguous online MS2 identification of unknown compounds of low-abundant individual CE(SDS) peaks is enabled.

Quantitative Proteome and Phosphoproteome Profiling in Magnaporthe oryzae.

The quality and consistency in every sample preparation procedure is crucial for any scientific output. Therefore, it is of utmost importance to have easy, economic, and robust sample preparation protocols. Here, we describe a simple and robust bottom-up proteomic sample preparation strategy for identification and label-free quantification (LFQ) of proteins and phosphoproteins. The presented workflow is designed for large-scale application and involves easy scalable and well-known robust sample preparation techniques, such as cell lysis with SDS buffer under heat, protein precipitation using methanol/chloroform, tryptic digest, and commercially available TiO2 phosphopeptide enrichment kits. Over a sample set of 48 samples of only 200mg fungal mycelium each, we quantified a median of 2937 proteins after processing in the IsoQuant software. The median peptide count was 10 peptides per protein leading to a median 65% sequence coverage. In addition, we identified a median of 3324 phosphopeptides (corresponding to 998 phosphoproteins) with 4874 phosphosites per sample. Over all samples, we achieved a median phosphopeptide enrichment efficiency of 77%. The distribution of serine/threonine/tyrosine (S/T/Y) phosphosites was 78.1%/21.2%/0.6%.

Comparison of metaproteomics workflows for deciphering the functions of gut microbiota in an animal model of obesity

Metaproteomics has emerged as a new, revolutionary approach to study gut microbiota functionality, but the lack of consistent studies in this field due to the great complexity of samples has prompted to search new strategies to achieve better metaproteome characterization. Some steps in sample preparation and data analysis procedures are critical for obtaining accurate results, therefore protein extraction buffers, digestion procedures and fractionation steps were tested here. Initially, two lysis buffers were used to improve protein extraction, two common digestion protocols were compared, and fractionation processes were employed at both the peptide and protein levels. The combination of these procedures resulted in five different methodologies; SDS buffer, in-gel digestion and fractionation at the peptide level provided the best results. Finally, the metaproteomics workflow was tested in a real case study with obese rats, in which a metagenomics study was previously performed. Important differences in protein levels were observed between groups that were potentially related to the taxonomical family, indicating that functional processes are modulated by the microbiota. Therefore, in addition to the necessity of combining different metaomics approaches, an optimized metaproteomics workflow such as the presented in this study is required to obtain a better understanding of the microbiota function. SignificanceGut microbiota has emerged as an important factor with affects the health balance in host. To study its function new methodologies are necessary and the most appropriate one seems to be metaproteomics. The lack of studies in this field requires a deeply research in the most accurate workflow to better comprehend such complex samples. In this paper, five different methodologies have been compared, mainly in the most critical steps in classical proteomics and the methodology chosen was validated in a real case study in obese animals.

2D SDS PAGE in Combination with Western Blotting and Mass Spectrometry Is a Robust Method for Protein Analysis with Many Applications.

Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS PAGE) is a method that separates proteins according to their isoelectric points in the first dimension and molecular masses in the second dimension. Evidence is provided that 2D SDS PAGE is reproducible, robust and compatible with SDS in both dimensions including isoelectric focusing in tube gels, the first dimension. The 2D gel pattern of rat liver microsomes shows more detail and sharper spot outlines when dissolved in SDS buffer with heating than in urea buffer and is better yet when dissolved in a mixture of both buffers. Quantification of 60 proteins in rat liver cytosol over a wide range of pI and MW gave linear plots of spot density versus total protein for loads of 200, 400 and 600μg protein dissolved in SDS buffer and run in triplicate on 2D gels (Average R2=0.987). Examples of biomedical applications are provided in which 2D proteins of interest found by comparing stained or western blotted 2D gel patterns were identified by mass spectrometry (MS).

Rapid Protein Analysis and Western Blotting by Capillary Gel Electrophoresis

Capillary gel electrophoresis has been used since the early 1980s to separate analytes based upon their mass to charge ratio. However, optical limitations prevented high sensitivity instruments from being developed, limiting functionality. As manufacturing capabilities have improved the high‐throughput, hands‐off nature of parallel capillary gel electrophoresis has made it a desirable technique for analysis of DNA, RNA, and protein. Traditionally, protein analysis and Western blotting have been accomplished using SDS‐PAGE followed by staining of proteins in the gel or by downstream transfer of proteins to a blotting membrane, respectively. The trademark feature of these methods is their labor‐intensive nature with many hours or days passing between sample loading and results. Additionally, manual analysis prevents processing of large numbers of samples.Advanced Analytical Technologies, Inc, (AATI) has developed a high‐throughput capillary electrophoresis instrument capable of running 12 samples in parallel with analysis of a complete 96‐well plate without user intervention. This method relies on the rapid fluorescent labeling (~5 min hands on, 15 min total) of total protein sample, with a linear labeling dynamic range of 1 ng − 1000 ng protein input, followed by heat denaturing of the sample in an SDS buffer. The samples are then separated electrophoretically through a proprietary gel matrix (~30 min run time). Sizing of samples is provided using internal markers and comparison to a standardized ladder, allowing for sizing of samples from 14 kDa to greater than 200 kDa with an accuracy of ≤5% and precision of less ≤5%. Relative quantification of proteins is accomplished by comparing the corrected peak area of sample peaks with a precision of ≤10%. Absolute quantification of proteins can be accomplished through a calibration curve using the same protein as the unknown, as each unique protein species will label at varying efficiencies.Capillary gel electrophoresis coupled with total protein labeling can be used to replace traditional Western blots by analyzing and quantifying immunoprecipitations. Analysis of immunoprecipitations by SDS‐PAGE and Coomassie staining are limited due to sensitivity and the copious amounts of antibody present in the sample. By using an unlabeled antibody coupled with labeled total protein, the capillary electrophoresis instrument will only detect the labeled protein of interest isolated by the immunoprecipitation. This provides relative concentration and size information analogous to that provided by the traditional western in a fraction of the time. The AATI protocol eliminates signal bleeding which allows for a greater dynamic range than film. A pull down of endogenous GAPDH demonstrated a 3‐fold dynamic response (0.01 μg – 10 μg protein input).By harnessing capillary gel electrophoresis, AATI has developed a high sensitivity SDS‐PAGE/Western blot replacement with a 3‐fold dynamic range, allowing for rapid analysis of total and purified protein. Please contact the authors at AATI‐us.com if you have interest in this technology.Support or Funding InformationThis work was funded by Advanced Analytical Technologies, Inc.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Two efficient methods for isolation of high-quality genomic DNA from entomopathogenic fungi

Conventional and commercial methods for isolation of nucleic acids are available for fungal samples including entomopathogenic fungi (EPF). However, there is not a unique optimal method for all organisms. The cell wall structure and the wide range of secondary metabolites of EPF can broadly interfere with the efficiency of the DNA extraction protocol. This study compares three commercial protocols: DNeasy® Plant Mini Kit (Qiagen), Wizard® Genomic DNA Purification Kit (Promega), and Axygen™ Multisource Genomic DNA Miniprep Kit (Axygen) and three conventional methods based on different buffers: SDS, CTAB/PVPP, and CTAB/β-mercaptoethanol versus three cell lysis procedures: liquid nitrogen homogenization and two bead-beating materials (i.e., tungsten-carbide and stainless-steel) for four representative species of EPF (i.e., Beauveria bassiana, Hirsutella citriformis, Isaria javanica, and Metarhizium anisopliae). Liquid nitrogen homogenization combined with DNeasy® Plant Mini Kit (i.e., QN) or SDS buffer (i.e., SN) significantly improved the yield with a good purity (~1.8) and high integrity (>20,000 bp) of genomic DNA in contrast with other methods, also, these results were better when compared with the two bead-beating materials. The purified DNA was evaluated by PCR-based techniques: amplification of translation elongation factor 1-α (TEF) and two highly sensitive molecular markers (i.e., ISSR and AFLP) with reliable and reproducible results. Despite a variation in yield, purity, and integrity of extracted DNA across the four species of EPF with the different DNA extraction methods, the SN and QN protocols maintained a high-quality of DNA which is required for downstream molecular applications.

Inclusion of 2-Mercaptoethanol in Lysis Buffer Could Interfere with Isolation of High Molecular Weight DNA from Freshwater Microalgae.

2-mercaptoethanol (2-ME), alongside polyvinylpyrrolidone is commonly used in plant DNA extractions to deal with polyphenols, which could interfere with extraction and downstream applications. 2-ME is also commonly used to denature proteins and nucleases, especially RNAses. On the contrary, we found that the presence of 2-ME in lysis buffer interfered with DNA extraction from 12 strains of freshwater microalgae, resulting in DNA with poor integrity. We also found that the TNES-urea buffer, commonly used for preservation and DNA extraction from fish, appears as effective as the SDS and CTAB buffer for some microalgae strains. Results from our study suggests that the inclusion of 2-ME in DNA extraction protocols may be detrimental for isolation of good quality DNA from freshwater microalgae, and therefore recommend eliminating it or testing varying concentrations of 2-ME when developing species-specific extraction protocols for microalgae.

Addition of urea and thiourea to electrophoresis sample buffer improves efficiency of protein extraction from TCA/acetone-treated smooth muscle tissues for phos-tag SDS-PAGE.

Phosphorylation analysis by using phos-tag technique has been reported to be suitable for highly sensitive quantification of smooth muscle myosin regulatory light chain (LC20 ) phosphorylation. However, there is another factor that will affect the sensitivity of phosphorylation analysis, that is, protein extraction. Here, we optimized the conditions for total protein extraction out of trichloroacetic acid (TCA)-fixed tissues. Standard SDS sample buffer extracted less LC20 , actin and myosin phosphatase targeting subunit 1 (MYPT1) from TCA/acetone treated ciliary muscle strips. On the other hand, sample buffer containing urea and thiourea in addition to lithium dodecyl sulfate (LDS) or SDS extracted those proteins more efficiently, and thus increased the detection sensitivity up to 4-5fold. Phos-tag SDS-PAGE separated dephosphorylated and phosphorylated LC20 s extracted in LDS/urea/thiourea sample buffer to the same extent as those in standard SDS buffer. We have concluded that LDS (or SDS) /urea/thiourea sample buffer is suitable for highly sensitive phosphorylation analysis in smooth muscle, especially when it is treated with TCA/acetone.

Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis.

The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer.

Peptides from American alligator plasma are antimicrobial against multi-drug resistant bacterial pathogens including Acinetobacter baumannii.

BackgroundOur group has developed a new process for isolating and identifying novel cationic antimicrobial peptides from small amounts of biological samples. Previously, we identified several active antimicrobial peptides from 100 μl of plasma from Alligator mississippiensis. These peptides were found to have in vitro antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In this work, we further characterize three of the novel peptides discovered using this process: Apo5, Apo6, and A1P.ResultsWe examined the activity of these peptides against multi-drug resistant strains and clinical isolates of common human pathogens. We investigated their structural characteristics using circular dichroism and tested for membrane disruption and DNA binding. These peptides were found to have strong in vitro activity against multi-drug resistant and clinically isolated strains of S. aureus, Escherichia coli, P. aeruginosa, and Acinetobacter baumannii. Apo5 and Apo6, peptides derived from alligator apolipoprotein C-1, depolarized the bacterial membrane. A1P, a peptide from the serpin proteinase inhibitor, did not permeabilize membranes. Performing circular dichroism analysis, Apo5 and Apo6 were found to be predominantly helical in SDS and TFE buffer, while A1P has significantly different structures in phosphate buffer, SDS, and TFE. None of these peptides were found to be hemolytic to sheep red blood cells or significantly cytotoxic up to 100 μg/ml after 24 h exposure.ConclusionsOverall, we suggest that Apo5 and Apo6 have a different mode of action than A1P, and that all three peptides make promising candidates for the treatment of drug-resistant bacteria, such as A. baumannii.

Extraction of high quality genomic DNA from Madhuca longifolia (J. Koenig ex l.) J.F. macbr.

Madhuca longifolia commonly known as the Mahua tree is a medium to large sized deciduous tree distributed in India, Nepal and Sri Lanka. It is found growing wild and cultivated. The tree is considered as a kalpavriksha by the tribal who are forest dwellers and keenly conserve this tree. The flowers and fruits are edible and the flowers are fermented to prepare mahua drink which is an integral part of their cultural heritage. The seeds are good source of edible fats and used to extract oil. Several pharmacological activities are reported using various parts of the tree. Very little information exists on the molecular aspects of Madhuca longifolia which requires high quality DNA. Optimization of DNA extraction using CTAB and SDS buffer system was standardized. A protocol for extraction of high quality DNA from tree species which are rich in polyphenols is hereby discussed.

Protein extraction from Ca-alginate encapsulated plant material for comparative proteomic analysis

The extensive use of encapsulation material in biotechnology drove the need to develop analytical techniques for this type of material. This study focuses on the specific problems of protein extraction from Ca-alginate encapsulated plant material. Proteomics is one of the fast-developing analysis categories, specifically for stress resistance and developmental changes in plant material. Sample preparation is a critical step in a two-dimensional gel electrophoresis proteome approach and is essential for good results. The aim was to avoid preliminary manipulations and get good quality material for comparative proteome analysis technique 2DE. The phenol extraction method and the complex method with preliminary TCA precipitation, SDS buffer and phenol phase were compared with respect to the efficiency and quality of the resulting 2DE gel. The most appropriate method turned out to be the TCA/phenol method with the phenol fractioning technique adapted to the gentian cell suspension. It resulted in a high protein concentration and good quality sample that could be analyzed using the standard separation procedures of 2DE and spectrometric identification with high efficiency. The work presented here confirms the possibility of obtaining a sufficient protein sample for effective proteomic analysis from a small number of capsules.

Integrated system for extraction, purification, and digestion of membrane proteins.

An integrated system was developed for directly processing living cells into peptides of membrane proteins. Living cells were directly injected into the system and cracked in a capillary column by ultrasonic treatment. Owing to hydrophilicity for broken pieces of the cell membrane, the obtained membranes were retained in a well-designed bi-filter. While cytoplasm proteins were eluted from the bi-filter, the membranes were dissolved and protein released by flushing 4% SDS buffer through the bi-filter. The membrane proteins were subsequently transferred into a micro-reactor and covalently bound in the reactor for purification and digestion. As the system greatly simplified the whole pretreatment processes and minimized both sample loss and contamination, it could be used to analyze the membrane proteome samples of thousand-cell-scales with acceptable reliability and stability. We totally identified 1348 proteins from 5000 HepG2 cells, 615 of which were annotated as membrane proteins. In contrast, with conventional method, only 233 membrane proteins were identified. It is adequately demonstrated that the integrated system shows promising practicability for the membrane proteome analysis of small amount of cells.

Towards the understanding of end-use quality in intermediate wheatgrass (Thinopyrum intermedium): High-molecular-weight glutenin subunits, protein polymerization, and mixing characteristics

Intermediate wheatgrass (Thinopyrum intermedium; IWG), is a perennial crop that is well-known for providing good environmental services. As a perennial relative of wheat, IWG has good potential for development into a perennial grain crop. But the diversity and potential of the end-use quality of IWG grain are still unknown. Here, to understand the end-use quality of IWG, we investigated the variability among seed from 60 IWG genotypes in high-molecular-weight glutenin subunits (HMW-GS), protein polymerization, and mixing characteristics. IWG genotypes have high grain protein content, ranging from 16.4 to 23.6%. Of the total proteins, the percentage of polymeric proteins show a large range across IWG genotypes, varying from 9.2 to 25.1%. Many genotypes have limited amounts of polymeric proteins, but several genotypes have large amounts of polymeric proteins and promising mixing properties, comparable to common wheat, Triticum aestivum. The amount of protein polymers in the SDS buffer unextractable fraction are significantly correlated (r ≥ 0.76) with mixograph parameters. Furthermore, we observed that HMW-GS variants showed differential contribution to protein polymerization and mixograph parameters. But the composition of HMW-GS in IWG was contributed by both parents, which might bring challenges for the evaluation of grain quality of open-pollinated IWG genotypes.

A High-Efficiency Cellular Extraction System for Biological Proteomics.

Recent developments in quantitative high-resolution mass spectrometry have led to significant improvements in the sensitivity and specificity of the biochemical analyses of cellular reactions, protein-protein interactions, and small-molecule-drug discovery. These approaches depend on cellular proteome extraction that preserves native protein activities. Here, we systematically analyzed mechanical methods of cell lysis and physical protein extraction to identify those that maximize the extraction of cellular proteins while minimizing their denaturation. Cells were mechanically disrupted using Potter-Elvehjem homogenization, probe- or adaptive-focused acoustic sonication, and were in the presence of various detergents, including polyoxyethylene ethers and esters, glycosides, and zwitterions. Using fluorescence spectroscopy, biochemical assays, and mass spectrometry proteomics, we identified the combination of adaptive focused acoustic (AFA) sonication in the presence of a binary poloxamer-based mixture of octyl-β-glucoside and Pluronic F-127 to maximize the depth and yield of the proteome extraction while maintaining native protein activity. This binary poloxamer extraction system allowed for native proteome extraction comparable in coverage to the proteomes extracted using denaturing SDS or guanidine-containing buffers, including the efficient extraction of all major cellular organelles. This high-efficiency cellular extraction system should prove useful for a variety of cell biochemical studies, including structural and functional proteomics.

Myristoylation profiling in human cells and zebrafish.

Human cells (HEK 293, HeLa, MCF-7) and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC). This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaíno et al., 2014 Nat. Biotechnol., 32, 223–6) (PXD001863 and PXD001876) and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development׳ by Broncel et al., Angew. Chem. Int. Ed.