Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.

/pdf/modified-tca-acetone-precipitation-of-plant-proteins-for-6p43yrdncw.pdf

Modified TCA/acetone precipitation of plant proteins for proteomic analysis

Protein precipitation is a common front-end preparation strategy for proteome analysis, as well as other applications (e.g., protein depletion for small molecule analysis, bulk commercial preparation of protein). Highly variable conditions used to precipitate proteins, ranging in solvent type, strength, time, and temperature, reflect inconsistent and low recovery. As a consequence, incomplete proteome coverage diminishes the utility of precipitation for proteome sample preparation ahead of mass spectrometry. We herein investigate and optimize the conditions affecting protein recovery through precipitation using acetone at a defined ionic strength. By increasing the salt concentration and incubation temperature with 80% acetone, we show that rapid (2 min) precipitation provides consistently high protein recovery (98 ± 1%) of complex proteome extracts. Rapid precipitation is also applicable to isolate dilute proteins starting as low as 1 μg mL-1. Furthermore, analysis of the protein pellet by bottom-up mass spectrometry (MS) reveals unbiased recovery of all proteins with respect to molecular weight, isoelectric point (pI), and hydrophobicity. Our robust strategy to isolate proteins maximizes recovery and throughput, exploiting the analytical advantages of precipitation over alternative techniques. Data are available via ProteomeXchange with identifier PXD015674.

Rapid and Quantitative Protein Precipitation for Proteome Analysis by Mass Spectrometry

The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has stretched national testing capacities to breaking points in almost all countries of the world. The need to rapidly screen vast numbers of a country’s population in order to control the spread of the infection is paramount. However, the logistical requirement for reagent supply (and associated cost) of RT-PCR based testing (the current front-line test) have been hugely problematic. Mass spectrometry-based methods using swab and gargle samples have been reported with promise, but have not approached the task from a systematic analysis of the entire diagnostic process. Here, the pipeline from sample processing, the biological characteristics of the pathogen in human biofluid, the downstream bio- and physical-chemistry and the all-important data processing with clinical interpretation and reporting, are carefully compiled into a single high-throughput and reproducible rapid process. Utilizing MALDI-ToF mass spectrometric detection to viral envelope glycoproteins in a systems biology-multidisciplinary team approach, we have achieved a multifaceted clinical MALDI ToF MS screening test, primarily (but not limited to) SARS-CoV-2, with direct application to other future epidemics/pandemics that may arise. The clinical information generated not only includes SARS-CoV-2 coronavirus detection–(Spike protein fragments S1, S2b, S2a peaks), but other respiratory viral infections detected as well as an assessment of generalised oral upper respiratory immune response (elevated total Ig light chain peak) and a measure of the viral immune response (elevated intensity of IgA heavy chain peak). The advantages of the method include; (1) ease of sampling, (2) speed of analysis, and much reduced cost of testing. These features reveal the diagnostic utility of MALDI-ToF mass spectrometry as a powerful and economically attractive global solution

Development of a Clinical MALDI-ToF Mass Spectrometry Assay for SARS-CoV-2: Rational Design and Multi-Disciplinary Team Work

Protein extracts obtained from cells or tissues often need to remove interfering substances for preparing high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples, especially TCA/acetone precipitation and phenol extraction. TCA/acetone precipitation, as a common method, is thought to minimize protein degradation and proteases activity as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation relies on the completely pulverizing and repeatedly rinse of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, a prolonged incubation in TCA/acetone or acetone can lead to the modifications and degradation of proteins, and the precipitated proteins are more difficult to re-dissolve. We have described a modified TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for various plant tissues in proteomic analysis.

https://www.biorxiv.org/content/biorxiv/early/2018/12/01/382317.full.pdf

Modified TCA/acetone precipitation of proteins for proteomic analysis

Seed storage proteins play a fundamental role in plant reproduction and human nutrition. They accumulate during seed development as reserve material for germination and seedling growth and are a major source of dietary protein for human consumption. Storage proteins encompass multiple isoforms encoded by multi-gene families that undergo abundant glycosylations and phosphorylations. Two-dimensional electrophoresis (2-DE) is a proteomic tool especially suitable for the characterization of storage proteins because of their peculiar characteristics. In particular, storage proteins are soluble multimeric proteins highly represented in the seed proteome that contain polypeptides of molecular mass between 10 and 130 kDa. In addition, high-resolution profiles can be achieved by applying targeted 2-DE protocols. 2-DE coupled with mass spectrometry (MS) has traditionally been the methodology of choice in numerous studies on the biology of storage proteins in a wide diversity of plants. 2-DE-based reference maps have decisively contributed to the current state of our knowledge about storage proteins in multiple key aspects, including identification of isoforms and quantification of their relative abundance, identification of phosphorylated isoforms and assessment of their phosphorylation status, and dynamic changes of isoforms during seed development and germination both qualitatively and quantitatively. These advances have translated into relevant information about meaningful traits in seed breeding such as protein quality, longevity, gluten and allergen content, stress response and antifungal, antibacterial, and insect susceptibility. This review addresses progress on the biology of storage proteins and application areas in seed breeding using 2-DE-based maps.

https://www.mdpi.com/1420-3049/23/10/2462/pdf

Advances in the Biology of Seed and Vegetative Storage Proteins Based on Two-Dimensional Electrophoresis Coupled to Mass Spectrometry

Maize is one of the most widely cultivated crops. It accumulates a large quantity of seed storage proteins, which are important for seed development and germination, and contribute to the nutritional quality of seeds. Based on solubility, the storage proteins are divided into albumins (water-soluble), globulins (salt-soluble), prolamins (alcohol-soluble), and glutelins (acid- or alkali-soluble). Maize hybrids are cultivated due to the superior performance of F1 hybrids than that of their parents, a phenomenon known as heterosis. However, the accumulation patterns of seed storage proteins in maize embryos between the hybrids and their parental inbred lines have not been compared. In the present study, two elite inbred lines of China, Zheng 58 and Chang 7-2, and their reciprocal hybrids (Zheng 58 × Chang 7-2 and Chang 7-2 × Zheng 58) were used to explore parental influences on the accumulation patterns of seed storage proteins in maize embryos. For this purpose, we focused on seed salt-soluble proteins (SSPs) in our experiments. The SSPs were selectively extracted from maize mature embryos after extensive removal of water-soluble albumin and separated using two-dimensional gel electrophoresis (2-DE), followed by mass spectrometry analysis. Our results indicated that the 2-DE SSP profiles of hybrids closely resembled those of their maternal parent rather than the paternal parent. In other words, 2-DE SSP profiles of Zheng 58 × Chang 7-2 were more similar those of Zheng 58 whereas such profiles of Chang 7-2 × Zheng 58 were more similar to those of Chang 7-2 although the 2-DE profiles of all four maize types were quite similar. In total, 12 relatively abundant SSPs spots representing five kinds of proteins were identified, of which nine protein spots displayed non-additive accumulation in at least one hybrid. This study provided additional data on dominance and partial dominance effects on maize hybrids embryos. Besides, earlier studies on accumulation profiles of globulin-1 (also known as vicilin), which is one of the most abundant globulins in maize embryos, also support the above results. This study would be helpful in revealing the mechanisms underlying SSPs accumulation patterns in the hybrids.

/pdf/accumulation-profiles-of-embryonic-salt-soluble-proteins-in-10x65p5lch.pdf

Accumulation Profiles of Embryonic Salt-Soluble Proteins in Maize Hybrids and Parental Lines Indicate Matroclinous Inheritance: A Proteomic Analysis.

/pdf/correction-modified-tca-acetone-precipitation-of-plant-t58azcvsmy.pdf

Correction: Modified TCA/acetone precipitation of plant proteins for proteomic analysis

As the complexity and concealment of network attacks are increasing day by day, construction of a comprehensive and effective defense system calls for modeling and formal description of the scenes, techniques and processes of network attacks. Integrating MITRE ATT&CK and MAL (Meta Attack Language) can be a good orientation for current research on network attack description method. This paper proposes a formal description method for network attacks, in which the assets in an information system and the attackers' techniques summarized in the ATT&CK Matrix serve as the vocabulary and an extended MAL serves as the syntax. First, this method differentiates various instances of the same asset category and involves formal description of the attack scene, including the hosts, network environment and their configurations. Second, this method uses a more reasonable classification system for the assets of an information system and a more simplified set of MAL symbols. As is verified by our experiment, this method can generate a comprehensive and clear formal description for the attack scene, attack techniques and attack process of a network attack on a real-world information system, which brings the description level of this kind of integrated method from attacker strategies to network nodes, and can be a guide to defense construction for a real-world information system.

Hang Zhang

Papers

Modified TCA/acetone precipitation of plant proteins for proteomic analysis

Modified TCA/acetone precipitation of proteins for proteomic analysis

Accumulation Profiles of Embryonic Salt-Soluble Proteins in Maize Hybrids and Parental Lines Indicate Matroclinous Inheritance: A Proteomic Analysis.

Correction: Modified TCA/acetone precipitation of plant proteins for proteomic analysis

An Integrated Formal Description Method for Network Attacks